• Title/Summary/Keyword: restriction-modification

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Cloning and Expression of the Bdi Methylase Gene in E. coli (대장균 내에서의 Bdi I Methylase 유전자의 클로닝과 발현)

  • 전희숙;김용석;최경래;노현모
    • Korean Journal of Microbiology
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    • v.25 no.1
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    • pp.40-45
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    • 1987
  • The gene for the Bdi I modification enzyme, which is one of Bdi I restriction-modification system, fromBrevibacterium divaricatum FERM 5948 was cloned and expressed in E. coli. For cloning of the Bdi I methylase gene, we have initially used three cloning site(EcoRI, BamHI and Sal I) of plasmid vector pBR 322 and adopted the retransformation method after Bdi I restriction endonuclease cleavage. Selection of transformants carrying the gene was based on the resistance of the modified plasmid encoding the enzyme to cleavage by Bdi I restriction enzyme, and the recombinant plasmid pBDIM 116 containing 5.6kb EcoRI insery was proved to carry the gene. Crude cell extracts prepared from strains carrying the plasmid pBDIM 116 contained an S-adenosylmethionine-dependent methyltransferase activity specific for the Bdi I recognition site, ATCGAT. The restriction map was constructed with 11 restriction enzyme, and the Bdi I restriction-modification system was also discussed.

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A Rapid Local Modification Algorithm for NURBS surface (NURBS 곡면의 고속 부분 수정 알고리듬 개발)

  • 김호찬;최홍태;김준안;이석희
    • Proceedings of the Korean Society of Precision Engineering Conference
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    • 2004.10a
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    • pp.1402-1405
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    • 2004
  • Algorithms on modification of NURBS surface requires modeling history to change its boundary conditions. The history is stored when the surface is modeled and saved in the corresponding model file. But when the model is transferred to other systems the history generally cannot be recognized. So modification algorithms without history is highly required. Previous works on the field is concentrated in the point based modification without any restriction condition. Therefore this study is intended to develope a curved based modification algorithm with restriction conditions. A rapid modification algorithm is suggested, implemented and tested.

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The Characterization of Mitochondrial DNA of Korean Ginseng (Panax ginseng C.A. Meyer) (고려인삼의 미토콘드리아 DNA의 분자생물학적 특성연구)

  • Lim, Yong-Pyo;Park, Kwang-Tae
    • Journal of Ginseng Research
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    • v.14 no.2
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    • pp.310-316
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    • 1990
  • This study was focused on the characterization of mitochondrial DNA (mtDNA) for molecular 9enetical approach of energy Production related mechanism in Panax ginseng. The simple and efficient method of mtDNA isolation from ginseng has been developed by modification of recently advanced methods. This procedure can successfully apply to mtDNA isolation of several plants. mtDNA of etiolated shoot and one-year root were digested with restriction endonucleases, but that of 6-year root not. Any difference was not observed in the restriction endonuclease digestion patterns among the ginseng variants. Molecular size of ginseng mtDNA was estimated at least 159 kb by the restriction endonuclease fragment analysis. The 4.5 kb extra band at the lane of EcoRII treatment could be observed in restriction patterns digested with the methylation sensitive endonucleases, BstN I and EcoRII. For construction of mitochondrial genomic library of ginseng, mtDNA was partially digested with EcoRl, and packaged with EMBL4 phage vector. Genomic library was screened and purified for further research including restriction mapping of ginseng mtDNA, and cloning of the genes. The gene of ATP synthase A subunit was cloned from the purified EMBL4 library clone No. 16. Now, clone No. 16 is subcloned for structure gene sequence analysis.

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Unbalanced Restriction Impairs SOS-induced DNA Repair Effects

  • Katna, Anna;Boratynski, Robert;Furmanek-Blaszk, Beata;Zolcinska, Natalia;Sektas, Marian
    • Journal of Microbiology and Biotechnology
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    • v.20 no.1
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    • pp.30-38
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    • 2010
  • The contribution of a type II restriction-modification system (R-M system) to genome integrity and cell viability was investigated. We established experimental conditions that enabled the achievement of hemimethylated and unmethylated states for the specific bases of the recognition sequences of the host's DNA. To achieve this, we constructed the MboII R-M system containing only one (i.e., M2.MboII) out of two functional MboII methyltransferases found in Moraxella bovis. Using the incomplete R-M system, we were able to perturb the balance between methylation and restriction in an inducible manner. We demonstrate that upon the SOS-induced DNA repair in mitomycin C treated cells, restriction significantly reduces cell viability. Similar results for the well-studied wild-type EcoRI R-M system, expressed constitutively in Escherichia coli, were obtained. Our data provide further insights into the benefits and disadvantages of maintaining of a type II R-M system, highlighting its impact on host cell fitness.

Anti-inflammatory Action of Calorie Restriction for Life-Prolongation: A Possible Mechanism

  • Chung, Hae-Young
    • Proceedings of the PSK Conference
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    • 2002.10a
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    • pp.173-174
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    • 2002
  • Oxidative modification of cellular structures and functions by redox imbalance is the basis of the current oxidative stress hypothesis of aging. The experimental support for this hypothsis has been generated from recent molecular probing on the interrelation between the age-related functional impairments and the pathogenesis. (omitted)

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The Characterization of Mitochondrial DNA of Korean Ginseng (Panax ginseng C.A. Meyer)

  • Lim, Yong-Pyo;Park, Kwang-Tae
    • Proceedings of the Ginseng society Conference
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    • 1990.06a
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    • pp.168-174
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    • 1990
  • This study was focused on the characterization of mitochondrial DNA (mtDNA) for molecular genetically approach of energy Production related mechanism in Panax Ein.fend. The simple and efficient method of mtDNA isolation from ginseng has been developed by modification of recently advanced methods. This procedure can successfully apply to mtDNA isolation of several plants. MtDNA of etiolated shoot and one-year root were digested with restriction endonucleases, but that of 6-year root not Any difference was not observed in the restriction endonuclease digestion patterns among the ginseng variants. Molecular size of ginseng mtDNA was estimated at least 159 kb by the restriction endonuclease fragment analysis. The 4.5 kb extra band at the lane of EcoRll treatment could be observed in restriction patterns digested with the methylation sensitive endonucleases, BstN 1 and EcoRll. For construction of mitochondrial genomic library of ginseng, mtDNA was partially digested with EcoRl, and packaged with EMBL4 phage vector Genomic library was screened and purified for further research including restricttion mapping of ginseng mtDNA, and cloning of the genes. The gene of ATP synthase A subunit was cloned koto the purified EMBL4 library clone No. 16. Now, clone No. 16 is subcloned for structure gene sequence analysis.

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Comparison of Gene Coding Clones Content in In vivo and In vitro Methyl-Filtration Libraries of Maize(Zea may)

  • Lee, Myung-Chul;Wing, Rod A;Suh, Seok-Cheol;Eun, Moo-Young
    • Korean Journal of Plant Resources
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    • v.20 no.6
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    • pp.491-498
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    • 2007
  • It has been hypothesized that efficient exclusion of methylated retrotransposons and repeated DNA region is one of the rapid and cost-effective approaches for comprehensive gene discovery in large genome size of maize. Three kinds of methylation-sensitive restriction enzymes, HapII, MspI and McrBC, were used to identify the restriction frequency of cytosine methylation sites in maize genome. Roughly 60% of total maize genomic DNA was restricted less than 500bp by McrBC, and the most of restricted small size fraction was composed retrotransposon. In order to validate the efficient construction of gene-rich shotgun library, we compare two gene-rich methyl-filtration shotgun libraries using in vivo and in vitro methyl-filtration system. The size selected DNA fraction by Sau3A-McrBC enzyme treated was very stable and has not appeared modification in E. coli, but most insert DNA size of partially digested with Sau3A were decrease less than 500bp by bacterial methylation-modification system. In compare of retroelements portion, A 44.6% of the sequences were retroelement in unmethyl-filtered library, and the most of them was Copia type, such as Prem, Opie and Ji. The portion of retroelement was drastically decreased to 25% and 20% by in vivo and in vitro filtration system, respectively.

Improvement of Transformation Efficiency Through In Vitro Methylation and SacII Site Mutation of Plasmid Vector in Bifidobacterium longum MG1

  • Kim, Jin-Yong;Wang, Yan;Park, Myeong-Soo;Ji, Geun-Eog
    • Journal of Microbiology and Biotechnology
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    • v.20 no.6
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    • pp.1022-1026
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    • 2010
  • The different cleavage patterns of pYBamy59 plasmid isolated from E. coli $DH5{\alpha}$ and B. longum MG1 by the cell extract of B. longum MG1 suggested that the main reason for its low transformation efficiency was related to the restriction modification (R-M) system. To confirm the correlation between the R-M system and transformation efficiency, in vitro methylation and site-directed mutagenesis were performed in pYBamy59. Sequence analysis of pYBamy59 fragments digested by the cell extract of B. longum MG1 revealed that all fragments were generated by restriction of the sequence recognized by SacII endonuclease. When pYBamy59 from E. coli was methylated in vitro by CpG or GpC methyltransferase, it was protected from SacII digestion. Site-directed mutagenesis, which removed SacII sites from pYBamy59, or in vitro methylation of pYBamy59 showed 8- to 15-fold increases in the transformation efficiency over intact pYBamy59. Modification of the SacII-related R-M system in B. longum MG1 and in vitro methylation in pYBamy 59 can improve the transformation efficiency in this strain. The results showed that the R-M system is a factor to limit introduction of exogenous DNA, and in vitro modification is a convenient method to overcome the barrier of the R-M system for transformation.

Modification of nutrition strategy for improvement of postnatal growth in very low birth weight infants

  • Choi, Ah Young;Lee, Yong Wook;Chang, Mea-young
    • Clinical and Experimental Pediatrics
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    • v.59 no.4
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    • pp.165-173
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    • 2016
  • Purpose: To identify the effects of modified parenteral nutrition (PN) and enteral nutrition (EN) regimens on the growth of very low birth weight (VLBW) infants. Methods: The study included VLBW infants weighing <1,500 g, admitted to Chungnam National University Hospital between October 2010 and April 2014, who were alive at the time of discharge. Subjects were divided according to 3 periods: period 1 (n=37); prior to the PN and EN regimen being modified, period 2 (n=50); following the PN-only regimen modification, period 3 (n=37); following both PN and EN regimen modification. The modified PN regimen provided 3 g/kg/day of protein and 1 g/kg/day of lipid on the first day of life. The modified EN regimen provided 3.5-4.5 g/kg/day of protein and 150 kcal/kg/day of energy. We investigated growth rate, anthropometric measurements at 40 weeks postconceptional age (PCA) and the incidence of extrauterine growth restriction (EUGR) at 40 weeks PCA. Results: Across the 3 periods, clinical characteristics, including gestational age, anthropometric measurements at birth, multiple births, sex, Apgar score, surfactant use and PDA treatment, were similar. Growth rates for weight and height, from time of full enteral feeding to 40 weeks PCA, were higher in period 3. Anthropometric measurements at 40 weeks PCA were greatest in period 3. Incidence of weight, height and head circumference EUGR at 40 weeks PCA decreased in period 3. Conclusion: Beginning PN earlier, with a greater supply of protein and energy during PN and EN, is advantageous for postnatal growth in VLBW infants.

Optimal condition for efficient DNA transfer in filamentous cyanobacteria by electroporation

  • Poo, Ha-Ryoung
    • Journal of Microbiology
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    • v.35 no.3
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    • pp.181-187
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    • 1997
  • Filamentous cyanobacteria are an ecologically important group of bacteria because they are able to provide both organic carbon fixed nitrogen that can support the nutritional requirements for other microorganisms. Because of their prokaryotic nature, they can also be used as potentially powerful model systems for the analysis of oxygenic photosynthesis and nitrogen fixation. Gene transfer is an indispensable procedure for genetic analysis of filamentous cyanobacteria. Electroporation was used to introduce foreign DNA into cyanobacterial cells. In experiments designed to optimize the electroporation technique, the effects of the field strength (amplitude of pulse) and time constant (duration of pulse), DNA concentration and host restriction/modification of DNA on the efficiency of electro-transformation were investigated. The results of this research revelaed that a high voltage pulse of short duration was effective for the electro-transformation of Anabaene sp. M131. The maximal number of transformants was obtained at 6 kV/cm with a pulse duration of 5 msec. The efficiency of electro-transformation was also sensitive to concenetration of DNA; even small amounts of DNA (0.01 .mu.g/ml) were able to gie a large number of transformants (1.0 * 10$\^$3/ cfu/ml).

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