• 제목/요약/키워드: polymerase

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한국 인삼 성분들에 관한 생화학적 연구(II) 인삼 성분들이 RNA 중합효소의 활동성에 미치는 영향 (Biochemical Studies on the Chemical Components of Borean Ginseng (ll) Effects of Ginseng Components on the Activity of RNA Polymerase)

  • 장세희;박인원
    • Journal of Ginseng Research
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    • 제1권1호
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    • pp.25-28
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    • 1976
  • 몇 가지 유기용매를 사용하여 인삼 추출액을 분별 분리하여 몇 개의 분칠들을 얻어서 이들이 RNA중합효소의 활동성에 미치는 영향을 조사하였다. 백삼과 홍삼에서 모두 RNA 중합효소의 활동성에 양성적인 효과를 가진 분획들을 얻었다. 백삼의 경우 RNA 중합효소에 양성적인 효과를 미치는 성분들은 전체 메탄을 추출액 분획, 초산에틸 추 출모액 및 이 모액의 수용성 분획들에서 확인되었고 흥삼의 경우에는 전체 메탄을 추출액 분획과 에테르 분획들에서 확인되었다. 이 사실은 중합효소에 양성적인 효과를 나타내는 성분들이 무극성 및 극성 부분들로 이루어져 있으며, 그들이 홍삼의 가공과 정에서 두 부분으로 분해되었을 것이라는 것을 암시의 준다.

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Increased DNA Polymerase Fidelity of the Lamivudine Resistant Variants of Human Hepatitis B Virus DNA Polymerase

  • Hong, Young-Bin;Choi, Yong-Wook;Jung, Gu-Hung
    • BMB Reports
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    • 제37권2호
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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.

Expression of Human Mitochondiral Aldehyde Dehydrogenase 2 in Mammalian Cells using Vaccinia Virus-T7 RNA Polymerase

  • Kang, Su-Min;Yoo, Seung-Ku;Lee, Ki-Hwan
    • Journal of Microbiology
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    • 제37권1호
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    • pp.41-44
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    • 1999
  • Human mitochondrial aldehyde dehydrogenase 2 (ALDH2) is mainly responsible for oxidation of acetaldehyde generated during alcohol oxidation in vivo. A full-length cDNA of human liver ALDH2 was successfully expressed using a vaccinia virus-T7 RNA polymerase system. The expressed ALDH2 had an enzymatic activity as high as the native human liver ALDH2 enzyme.

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Genetic Similarity and Diversity in Crucian Carp(Carassius carassius) Populations by Polymerase Chain Reaction-Random Amplified Polymorphic DNAs

  • Yoon, Jong-Man;Kim, Tae-Sun;Kim, Jong-Yeon
    • 한국어업기술학회:학술대회논문집
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    • 한국어업기술학회 2001년도 춘계 수산관련학회 공동학술대회발표요지집
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    • pp.332-333
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    • 2001
  • Genomic DNA was extracted from the blood of the freshwater crucian carp(Carassius carassius) from Kunsan in Korea, representing genetic similarity by polymerase chain reaction amplification of DNA as twelve of arbitrary primers. The electrophoretic analysis of polymerase chain reaction-random amplified polymorphic DNAs(PCR-RADP) products showed the high levels of similarity between different individuals in crucian carp.

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The 52 kD Protein Gene of Odontoglossum Ringspot Virus Containing RNA-Dependent RNA Polymerase Motifs and Comparisons with Other Tobamoviruses

  • Park, Won-Mok
    • Journal of Plant Biology
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    • 제38권2호
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    • pp.129-136
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    • 1995
  • Complementary DNA of the genomic RNA of odontoglossum ringspot virus Cymbidium strain (ORSV-Cy) was synthesized from polyadenylated viral RNA and cloned. Selected clones containing the viral RNA-dependent RNA polymerase gene of the virus has been sequenced by automated sequencing system. The complete nucleotide sequence of an open reading frame is 1377 base pairs in length, and encodes a protein of 458 amino acids about 52, 334 D. The 52 kD protein of ORSV shares four sequence motifs characteristic of viral RNA-dependent RNA polymerase. Comparison of the ORSV 52 kD protein sequence with that of other five viruses in tobamovirus group showed 76.0 to 60.7% homologies at the amino acid level and the conservation of the four motifs betwen the viruses.

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Overexpression, Purification, and Characterization of the Herpes Simplex Virus-1 DNA Polymerase-UL42 Protein Complex

  • Song, Byeong-Doo;Lehman, I. Robert
    • BMB Reports
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    • 제31권6호
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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.

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Evaluation of Inhibitory Effects of Thiobarbituric Acid Derivatives Targeting HCV NS5B Polymerase

  • Lee, Jong-Ho;Lee, Sang-Yoon;Park, Mi-Young;Ha, Hyun-Joon;Myung, Hee-Joon
    • Journal of Microbiology and Biotechnology
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    • 제20권3호
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    • pp.510-512
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    • 2010
  • A series of thiobarbituric acid derivatives were constructed and evaluated for inhibitory activity on hepatitis C virus NS5B polymerase. In biochemical assays using purified viral polymerase and RNA template, the $IC_{50}$ value was improved to 0.41 ${\mu}M$ from the original compound's 1.7 ${\mu}M$ value. In HCV sub genomic replicon assay, the $EC_{50}$ value was improved to 3.7 ${\mu}M$ from the original compound's 12.3 ${\mu}M$ value. $CC_{50}$ was higher than 77 ${\mu}M$ for all compounds tested, suggesting that they are useful candidates for anti-HCV therapy.

Comparison of quantitative detection of periodontal pathogens before and after scaling by real-time polymerase chain reaction

  • Kim, Young-Sun;Lee, Jung-Hwa;Lee, Young-Eun
    • 한국치위생학회지
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    • 제15권6호
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    • pp.1063-1071
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    • 2015
  • Objectives: The purpose of the study is to investigate the quantitative detection of periodontal pathogens before and after scaling by real-time polymerase chain reaction. Methods: Participants were voluntarily recruited at D university, and saliva samples were extracted before and after scaling. Multiple real-time polymerase chain reactions were used to analyze characteristics and the amount of nine kinds of periodontal pathogens; Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Tannerella forsythia, Treponema denticola, Prevotella intermedia, Fusobacterium nucleatum, Parvimonas micra, Campylobacter rectus, and Eikenella corrodens. Results: After scaling, most periodontal pathogens except Eikenella corrodens were significantly decreased in all subjects(p<0.05). In addition, the percentage of microorganisms associated with disease, the microorganism risk index of periodontitis and the prevalence of red complex, orange complex, and Aggregatibacter actinomycetemcomitans was also significantly reduced after scaling(p<0.05). Conclusions: Scaling decreased in the amount of major periodontal pathogens and periodontitis prevalence rate.

A Simple Method for Elimination of False Positive Results in RT-PCR

  • Martel, Fatima;Grundemann, Dirk;Schomig, Edgar
    • BMB Reports
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    • 제35권2호
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    • pp.248-250
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    • 2002
  • Discrimination between the amplification of mRNA and contaminating genomic DNA is a common problem when performing a reverse transcriptase-polymerase chain reaction (RT-PCR). Even after treatment of the samples with DNAse, it is possible that negative controls (samples in which no reverse transcriptase was added) will give positive results. This indicates that there was amplification of DNA, which was not generated during the reverse transcriptase step. The possibility exists that Taq DNA polymerase acts as a reverse transcriptase, generating cDNA from RNA during the PCR step. In order to test this hypothesis, we incubated samples with a DNAse-free RNAse after the cDNA synthesis. Comparison of the results that were obtained from these samples (incubated with or without DNAse-free RNAse) confirms that the reverse transcriptase activity of Taq DNA polymerase I is a possible source of false positive results when performing RT-PCR from intronless genes. Moreover, we describe here a simple and rapid method to overcome the false positive results that originate by this activity of Taq polymerase.

T7 발현체계에서 chloramphenicol acetyltransferase의 선택적 과잉생산 (Selective overproduction of chloramphenicol acetyltransferase in the T7 expression system)

  • 김한복;강창원
    • 미생물학회지
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    • 제27권4호
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    • pp.317-322
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    • 1989
  • A gene can be selectively overexpressed in E. coli by utilizing the phage T7 RNA polymerase's stringent recognition and active transcription of the T7 promoter. The T7 expression system was constructed such that the T7 RNA polymerase gene is under the control of lacUV5 promoter in one plasmid, and that the target gene, the promoterless chloramphenicol acetyltransferase (CAT) gene with E. coli ribosome binding site is under the control of T7 promoter in the other plasmid. Only the E. coli cells containing both plasmids show high resistance to chloramphenicol. When the copy number of the runaway plasmid containing the polymerase gene was varied by a temperature shift, amounts of the CAT protein synthesized upon induction was correspondingly changed as shown in SDS gel electrophoresis.

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