• Microbiology and Biotechnology Letters
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• v.22 no.6
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• pp.577-583
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• 1994

To classify Lactobacillus casei strains on the basis of difference in their chromosomal DNA sequence, we have performed polymerase chain reactions on their chromosomal DNA by using random primers, and followed by analyzing randomly amplified polymorphic DNA fragments. We also developed a mini-preparative method to isolate PCR-grade chromosomal DNA from Lacto- bacillus casei strains within 3 hours. Based on RAPD pattems by polymerase chain reactions with degenerated random primers, 4 Lactobacillus casei strains and 2 mutant strains were successfully discriminated. Results were very sensitive, strain-specific and reproducible. It was also reliable. These results suggest that RAPD may be applied efficiently for the identification of several Lactoba- cillus casei strains.

• Journal of Microbiology and Biotechnology
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• v.18 no.8
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• pp.1377-1385
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• 2008

The uracil-sensing domain in archaeal family B-type DNA polymerases recognizes pro-mutagenic uracils in the DNA template, leading to stalling of DNA polymerases. Here, we describe our new findings regarding the molecular, mechanism underpinning the stalling of polymerases. We observed that two successive deaminated bases were required to stall TNA1 and KOD1 DNA polymerases, whereas a single deaminated base was enough for stalling Pfu DNA polymerase, in spite of the virtually identical uracil-sensing domains. TNA1 and KOD1 DNA polymerases have a much higher extension rate than Pfu DNA polymerase; decreasing the extension rate resulted in stalling by TNA1 and KOD1 DNA polymerases at a single deaminated base. These results strongly suggest that these polymerases require two factors to stop DNA polymerization at a single deaminated base: the presence of the uracil-sensing domain and a relatively slow extension rate.

• BMB Reports
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• v.31 no.6
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• pp.585-589
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• 1998

The herpes simplex virus type-1 (HSV-1)-encoded DNA polymerase consists of two subunits, the products of the UL30 and UL42 genes. UL30 and UL42 were coexpressed in Sf9 cells infected with recombinant baculoviruses carrying the two genes. The UL30 and UL42 gene products remained tightly associated throughout the purification, which led to a near homogeneous heterodimer composed of the DNA polymerase and UL42 protein. The DNA polymerase-UL42 protein heterodimer, purified from the recombinant baculovirus-infected Sf9 cells, showed the same high degree of processivity of deoxynucleotide polymerization as the enzyme purified from the HSV-1 infected primate cells. Like the latter, it contained a 3'-5' exonuclease activity that specifically hydrolyzes an incorrectly matched nucleotide at the 3' terminus of a primer, thereby contributing to the fidelity of DNA replication.

• The Korean Journal of Zoology
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• v.26 no.3
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• pp.171-179
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• 1983

Amounts of DNA single strand breaks and unscheduled DNA synthesis in CHO cells exposed to MMS were increased in the presence of 3-aminobenzamide, a potent inhibitor of poly (ADP-ribose) polymerase. However, those in cells irradiated with UV-light were decreased. These results suggest that poly (ADP-ribose) polymerase acts negatively on the MMS-induced base excision repair but positively on the UV-induced nucleotide excision repair. In the combined treatment with MMS and UV-light in the presence of this inhibitor, amounts of strand breaks were just the same as those in the absence of the inhibitor. But those of unscheduled DNA synthesis were increased up to the amount induced by UV-light alone. These results may suggest that poly (ADP-ribose) polymerase affects the incision step of excision repair induced by MMS and UV-light independently, and that it may potentiate the complete cleaving of UV-induced pyrimidine dimers possibly by the repair enzymes which might have been partially inactivated by MMS.

• Environmental Mutagens and Carcinogens
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• v.8 no.1
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• pp.1-12
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• 1988

The effects of aphidicolin (APC), an inhibitor of DNA polymerase alpha, or 2', 3'-dideoxythymidine 5'-triphosphate (ddTTP), an inhibitor of DNA polymerase beta, on the repair of DNA damage induced by ethyl methanesulfonate (EMS) or bleomycin (BLM) were investigated in Chinese hamster ovary (CHO)-K1 cells. Three assays were employed in this study: unscheduled DNA synthesis, alkaline elution and alkaline sucrose gradient sedimentation. It was shown that APC or ddTTP inhibited DNA induced by EMS, and thus, the post-treatment with APC or ddTTP following EMS treatment was resulted in the more amount of unscheduled DNA synthesis, and the more accumulation of DNA single-stand breaks than the cells post-incubated without APC or ddTTP. While, in the BLM induced DNA repair, only ddTTP inhibited DNA repair induced by BLM. And thus, the groups post-incubated with or without APC after BLM treatment had the same value in the amount of unscheduled DNA synthesis and of DNA single-strand breaks, while post-treatment with ddTTP was resulted in the increased amount of unscheduled DNA synthesis and the increased DNA sin -strand breaks than the group without ddTTP. These results suggested that both of DNA polymerase $\alpha$ and $\beta$ participated in the repair of DNA damage induced by EMS, but in BLM-induced DNA repair, polymerase $\beta$ participated.ipated.

• BMB Reports
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• v.37 no.2
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• pp.167-176
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• 2004

Although efficient antiviral lamivudine is used for HBV-infected patients, a prolonged treatment with nucleoside analogs often results in lamivudine-resistant variants. In this study, we evaluated the fidelity of the lamivudine-resistant variants. The FLAG-tagged wild-type (FPolE) and Met550 variants (FPolE/M550A, M550V, and M550I) of HBV DNA polymerases were expressed in insect cells then purified. Like many other reverse transcriptases, no $3'{\rightarrow}5'$ exonuclease activity was detected in the HBV DNA polymerase. Since there is no proofreading activity, then the use of the site-specific nucleotide misincorporation method is beneficial. From the $f_{ins}$ value analysis, it is evident that M550I and M550V exhibit higher fidelity values than the wild-type HBV DNA polymerase, while M550A exhibits similar fidelity values. It is therefore suggested that lamivudine resistance comes from the stringency to dNTP binding and the discrimination of dCTP and lamivudine in M550V and M550I.

• KSBB Journal
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• v.10 no.3
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• pp.264-270
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• 1995

Trigonopsis variabilis is a strong producer of D-amino acid oxidase that can transform cephalosporin C(ceph C) to ${\alpha}$-keto-adipyl-7-aminocephalosporanic acid(AKA-7ACA). Polymerase chain reaction (PCR) was applied to isolate the D-AAO gene from T. variabilis. To clone the PCR fragment, four different methods were examined using enzymatic reactions of Taq DNA polymerase, Klenow, T4 DNA polymerase I, Alkaline phosphatase Calf Intestinal, and T4 kinase. Ligation of phosphorylated blunt-end PCR fragment and dephosphorylated blunt-end of pUC18 plasmid yielded the best cloning efficiency One of recombinant E. coli transformants showed D-AAO activity against ceph C in both cell extracts and permeabilized cells.

• Environmental Mutagens and Carcinogens
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• v.9 no.1
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• pp.23-32
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• 1989

Chinese hamster ovary (CHO)-K1 cells echibited a differential sensitivity in the process of DNA repair synthesis induced by ethyl methanesulfonate (EMS) or bleomycin (BLM) in relation to cell cycle. Two assays were employed in this study: alkaline elution and unscheduled DNA synthesis. The post-treat-ment with aphidicolin (APC), an inhibitor of DNA polymerase alpha, inhibited DNA repair synthesis induced by EMS in G2 phase, while APC did not show any effect on BLM-induced DNA repair synthesis in all phases. On the other hands, the 2', 3'-dideoxythymidine (ddTTP), an inhibitor of DNA polymerase beta, inhibited DNA repair synthesis induced by EMS or BLM in both of G1 and G2 phases. These results suggested that the involvement of DNA polymerase alpha and beta in DNA repair was dependent on cell stage or used chemical agent.

• International Journal of Oral Biology
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• v.46 no.3
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• pp.140-145
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• 2021

This study aimed to develop Lautropia mirabilis-specific quantitative real-time polymerase chain reaction (qPCR) primers based on the sequence of DNA-directed RNA polymerase subunit beta gene. The PrimerSelect program was used in designing of the qPCR primers, RTLam-F4 and RTLam-R3. The specificity of the qPCR primers were performed by conventional PCR with 37 strains of 37 oral bacterial species, including L. mirabilis. The sensitivity of the primers was determined by qPCR with the serial dilution of purified genomic DNA of L. mirabilis KCOM 3484, ranged from 4 ng to 4 fg. The data showed that the qPCR primers could detect only L. mirabilis strains and as little as 40 fg of genome DNA of L. mirabilis KCOM 3484. These results indicate that this qPCR primer pair (RTLam-F4/RTLam-R3) may be useful for species-specific detection of L. mirabilis in epidemiological studies of oral bacterial infectious diseases such as periodontal disease.

• Journal of Korean Society of Forest Science
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• v.81 no.4
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• pp.320-324
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• 1992

It has been reported that there are two distinct phenotypes in Fraxinus mandshurica Rupr. growing in Korea. Recently developed polymerase chain reaction(PCR) was used to detect DNA sequence polymorphism in the species. Using a thermostable DNA polymerase and synthetic DNA primers, unknown DNA sequences from the species were randomly amplified. The two types of the species produced different DNA amplification pattern with three different primers tested. Although DNA polymorphism was detected among individuals within types, each type has its own distinct pattern. The two types could be easily differentiated by trier characteristic predominant bands.