• Title/Summary/Keyword: DNA Synthesis

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Construction and Analysis of a DNA Microarray for the Screening of Biosynthetic Genes of Secondary-Metabolites formation in Streptomyces (방선균 유래 이차대사 생합성 유전자 분석용 DNA Microarray 제작 및 해석)

  • Nam Soo Jung;Kang Dae-Kyung;Rhee Ki Hyeong;Kim Jong-Hee;Kang Sang Sun;Chang Yong Keun;Hong Soon-Kwang
    • Korean Journal of Microbiology
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    • v.41 no.2
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    • pp.105-111
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    • 2005
  • Streptomyces produces many kinds of secondary-metabolites including antibiotics. Screening of a new compound and elucidation of a biosynthetic pathway for the secondary metabolites are very important fields of biology, however, there is a main problem that most of the identified compounds are already researched compounds. To solve these problems, a microarray system that is based on the data related to the biosynthetic genes for secondary-metabolites was designed. For the main contents of DNA microarray, the important genes for the bio-synthesis of aminoglycosides, polyenes group, enediyne group, alpha-glucosidase inhibitors, glycopeptide group, and orthosomycin group were chosen. A DNA microarray with 69 genes that were involved in the bio-synthesis for the antibiotics mentioned above was prepared. The usability of the DNA microarray was confirmed with the chromosomal DNA and total RNA extracted from S. coelicolor whose genomic sequence had already been reported.

Biochemical Characterization of the Herpes Simplex Virus-1 DNA Polymerase

  • Song, Byeong-Doo;Lehman, I. Robert
    • BMB Reports
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    • v.32 no.5
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    • pp.492-496
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    • 1999
  • We have investigated the biochemical properties of the herpes simplex virus type 1 (HSV-1) DNA polymerase without the UL42 protein (Pol), purified from insect cells infected with a recombinant baculovirus containing the UL30 gene. BSA and DTT have inhibitory effects on dAMP incorporation. Pol showed a greater turnover rate of steady-state single nucleotide incorporation at 12 mM $MgCl_2$ than at 2 mM $MgCl_2$. However, it showed a greater processivity of DNA synthesis at lower $MgCl_2$ concentration (1 mM, 2 mM) than at a higher $MgCl_2$ concentration (12.5 mM). These results are consistent with a slow DNA dissociation at lower $MgCl_2$ concentrations. Pol does not incorporate a correct nucleotide into the primer with an incorrect nucleotide at the end; instead, it preferentially excises the incorrect nucleotide at the 3' end of the primer. Pol has DNA polymerase activity at pHs 6.5 and 7.5 but little at pHs 5.5, 8.5, and 9.5. It has exonuclease activity at pHs 6.5, 7.5, and 8.5 but little at pHs 4.5, 5.5, and 9.5. The finding that Pol has exonuclease activity but not DNA polymerase at pH 8.5 suggests that DNA binds to Pol, but deoxynucleotide binding or incorporation does not occur at pH 8.5.

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Complementary DNA Cloning and Restriction Mapping of Nuclear Inclusion Body and Coat Protein Genes of Turnip Mosaic Virus-Ca Strain Genomic RNA (순무모자이크 바이러스 Ca계통 핵봉입체와 외피단백질 유전자의 cDNA 클로닝 및 제한효소 지도작성)

  • 류기현;박원목
    • Korean Journal Plant Pathology
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    • v.10 no.3
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    • pp.235-239
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    • 1994
  • Viral RNA was extracted from purified Chinese cabbage strain of turnip mosaic virus (TuMV-Ca) from infected leaves of turnip. Polyadenylated genomic viral RNA was recovered by oligo (dT) cellulose column chromatography and used as a template for the synthesis of complementary DNA (cDNA). Recombinant plasmids contained cDNA ranged from about 900 bp to 2, 450 bp were synthesized. Among the selected 41 transformants, pTUCA31 and pTUCA35 had over 2 Kbp cDNA insert. Restriction endonuclease patterns of the clones examined were very similar among them. Clones pTUCA23 and pTUCA31 were overlapped with pTUA35. The longest clone pTUCA35, encoding 3'-end, showed that it contained two sites for EcoRI, and one site for BamHI, ClaI, HincII, SacI and XbaI, respectively. The restriction mapping indicated that the clone pTUCA35 contained partial nuclear inclusion body gene, complete coding region of the coat protein and 3' untranslated region of TuMV-Ca genomic RNA.

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Genetic Effects of Pesticides in the Mammalian Cells: II. Mutagenesis in L5178Y Cells and DNA Repair Induction

  • Park, sang-Gi;Lee, Se-Yong
    • The Korean Journal of Zoology
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    • v.20 no.4
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    • pp.159-168
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    • 1977
  • In order to evaluate the mutagenic potentential in mammalian system for those pesticides which were proved to be mutagenic in Salmonella microsome assay system, we have studied drug-resistant mutagenesis in cultured L5178Y cells and unscheduled DNA synthesis in human lymphocytes in vitro. We have tested five pesticides: insecticides DDVP and trichlorfon, fungicide TMTD and herbicides MO and NIP. Of these pesticides, TMTD induced weak mutation to MTX-resistance in L5178Y cells in vitro and gave positive responses in DNA repair assay system. Therefore, its potential genetic risks in human beings should be re-evaluated. DDVP and trichlorfon gave negative response in L5178Y mutagenesis test system but stimulated incorporation of $^{3}H$-TdR in DNA repar assay. MO and NIP gave also negative responses both in L5178Y mutagenesis test systemand in DNA repair assay system.

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Effects of Ginseng Radix Against Osteoblasts Damaged by Endocrine Disrupting Chemicals (환경호르몬에 의해 손상된 골모세포에 대한 인삼의 영향)

  • Hong Gi Youn;Park Seung Taeck;Jeon Byung Hun;Seo Eun A
    • Journal of Physiology & Pathology in Korean Medicine
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    • v.16 no.2
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    • pp.338-342
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    • 2002
  • In order to evaluate the effect of Ginseng Radix(GR) against endocrine disrupting chemicals(EDC), cultured mouse osteoblasts were preincubated with various concentrations of GR extract before the exposure of Bisphenol A for 12 hours. Cytotoxic effect of Bisphenol A was measured by the XTT assay. In addition, the protective effect of GR over Bisphenol A-induced cytotoxicity on osteoblasts was assessed by the DNA and protein synthesis in these cultures. The results were as follows : Osteoblastic cell viability was decreased in dose and time dependent manner after exposed to various concentrations of Bisphenol A. Midcytotoxicity value(MCV50) of Bisphenol A was determined at 6μM Bisphenol A after osteoblasts were grown for 12 hours in the media containing various concentrations of Bisphenol A. Amount of DNA synthesis was increased in dose-dependent manner after cultured osteblasts were pretreated with GR for 2hrs before exposure to Bisphenol A for 12 hours. Amount of protein synthesis was increased in dose-dependent manner after cultured osteoblasts were pretreated with GR for 2 hours before exposure of Bisphenol A for 12 hours. From these results, it is suggested that Bisphenol A was highly toxic by the decrease of the cell viability, and GR is effective in the prevention of Bisphenol A-induced cytotoxicity by the increase of DNA and protein syntheses in cultured mouse osteoblasts.

Effects of three kinds of Radix Rehmanniae Water Extract in Cultured Rat Myocardial Cells (삼종 지황 추출물이 배양 심근세포에 미치는 영향)

  • Hwang In Jin;Kwon Kang Beom;Cho Hyun Ik;Min Young Gi;Heo Jae Hyuk;Kim Gu Hwan;Ryu Do Gon
    • Journal of Physiology & Pathology in Korean Medicine
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    • v.16 no.6
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    • pp.1117-1121
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    • 2002
  • To test the protective effect of herbal medicine on myocardial damage against oxygen free radical-induced myocardiotoxicity, cytotoxicity was examined using MTT, Beating rate and DNA synthesis assay in the presence of water extract of three kinds of Radix Rehmanniae. Myocardial toxicity was evaluated in neonatal rat myocardiocytes in cultures. The results of these experiments were obtained as follows : Xanthine oxydase/hypoxanthine resulted in a decrease in viability, beating rate and DNA synthesis in cultured myocardial cells. Radix Rehmanniae Recens(生地黃, RRR) water extract shows effects of protection from the cardiocyte toxicity induced by xanthine oxydase/hypoxanthine treatment such as increases in beating rate. Radix Rehmanniae Preparat(熟地黃, RRP) water extract shows effects of protection from the cardiocyte toxicity induced by xanthine oxydase/hypoxanthine treatment such as increases in DNA synthesis. These results show that xanthine oxydase/hypoxanthine elicits toxic effects in cultured myocardial cells derived from neonatal rat, and suggest that water extract of three kinds of Radix Rehmanniae is very effective in the prevention of xanthine oxydase/hypoxanthine-induced cardiotoxicity.

The Anti-Proliferation Effects and Its Mechanism of Bupleurum falcatum on Human Mesangial Cell (시호의 사구체 메산지움 세포 증식억제 효능 및 작용기전 연구)

  • Lee, Byung-Cheol;Ahn, Young-Min;Doo, Ho-Kyung;Ahn, Se-Young
    • The Journal of Internal Korean Medicine
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    • v.25 no.4
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    • pp.9-17
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    • 2004
  • Objective : Mesangial cell proliferation and excessive accumulation of extracellular matrix (ECM) proteins is the common pathologic feature of glomerulosclerosis, and platelet-derived growth factor (PDGF) BB-chain, transforming growth factor betal $(TGF-{\beta}1)$, cyclin dependent kinases (CDK) and CDK inhibitors mediated in these pathophysiological processes. Bupleurum falcatum which is one of the most widely used components in traditional oriental medicines, has multiple pharmacological effects, such as antipyretic, analgesic, immune modulating, anti-inflammatory, anti-allergic, anti-thrombotic, anti-atherosclerotic, and antitussive effects. Methods : In this study, we evaluated the influence of Bupleurum falcatum on mesangial cell proliferation, DNA synthesis and expression of PDGF-BB chain, $TGF-{\beta}1$, CDKI, CDK2, CDK4, p21 and p27 in fetal bovine serum (FBS)-activated human mesangial cell. Results : Bupleurum falcatum reduced the mesangial cell proliferation and DNA synthesis more than control and captopril. And in the ELISA analysis of $TGF-{\beta}1$, and RT-PCR of PDGF-BB chain, CDK1, CDK2, CDK4, p21, and p27, Bupleurum falcatum inhibited the expression of $TGF-{\beta}1$ protein and PDGF-BB, CDK1, CDK2 gene and promoted that of p21 gene in a dose-dependent manner in comparing with control and captopril. Conclusions: These results suggest that Bupleurum falcatum may inhibit the mesangial cell proliferation and DNA synthesis by regulation of PDGF-BB and $TGF-{\beta}1$ expressions, and by modulation of CDK1, CDK2 and p21 expression.

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Inhibitory Effects of Kimchi Extracts on the Growth and DNA Synthesis of Human Cancer Cells

  • Hur, young-Mi;Kim, So-Hee;Park, Kun-Young
    • Preventive Nutrition and Food Science
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    • v.4 no.2
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    • pp.107-112
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    • 1999
  • Effect of solvent extracts and juice supernatants from kimchis on the growth of various human cancer cells was studied, comparing with the actions on the normal cells. Inhibitory effect of kimchi extracts on[3H] thymidine incorporation n cancer cells was also investigated. The methanol extract, hexane extract and methanol soluble fraction (MSF) of 3-week fermented kimchi did not have growth inhibitory effect on Ac2F rat normal liver cells at the concentrations of 0.5~2%. However, marked decrease in the growth of AGS human gastric cancer cells was shown by the treatment of those extacts. The juice from the kimchi samples also suppressed the growth of K-562 human leukemia cells and MG-63 human osteosarcoma cells. Especially, the juice of 3-week fermented kimchi exhibited the strong growth inhibitory effect in MG-63 human osteosarcoma cells. At the photomicrographs, growth inhibition and morphological change of the cells treated with kimchi juice were observed. And the solvent extracts of 3-week fermented kimchi suppressed the growth of cancer morethan the extracts or juices from fresh and 6-week fermented kimchi. When AGS human gastric cancer cels were treated with the extracts of 3-week fermented kimchi, [3H] thymidine incorporation in the cells also decreased. These results showed that kimchi extracts and juices had growth inhibitory effects on human osteosarcoma, leukemia and gastric cancer cells, but had no toxicity to the normal cells. We suggest that kimchi might have anticancer effect in part due to inhibition of the growth and DNA synthesis of cancer cells.

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