• Title/Summary/Keyword: Covalently linked

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A Proteomic Approach to Study msDNA Function in Escherichia coli

  • Jeong, Mi-Ae;Lim, Dongbin
    • Journal of Microbiology
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    • v.42 no.3
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    • pp.200-204
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    • 2004
  • Retron is a prokaryotic genetic element that produces multicopy single-stranded DNA covalently linked to RNA (msDNA) by a reverse transcriptase. It was found that cells producing a large amount of msDNA, rather than those that did not, showed a higher rate of mutation. In order to understand the molecular mechanism connecting msDNA production to the high mutation rate the protein patterns were compared by two dimensional gel electrophoresis. Ten proteins were found to be differentially expressed at levels more than three fold greater in cells with than without msDNA, nine of which were identified by MALDI TOF MS. Eight of the nine identified proteins were repressed in msDNA-producing cells and, surprisingly, most were proteins functioning in the dissimilation of various carbon sources. One protein was induced four fold greater in the msDNA producing cells and was identified as a 30S ribosomal protein S2 involved in the regulation of translation. The molecular mechanism underlying the elevated mutation in msDNA-producing cell still remains elusive.

Scintillation Proximity Assay (섬광근접측정법)

  • Choi, Chang-Woon
    • The Korean Journal of Nuclear Medicine
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    • v.33 no.6
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    • pp.461-465
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    • 1999
  • Scintillation proximity assay (SPA) is a unique type of radioimmunoassay and makes it possible to use radioisotopes for monitoring binding reactions continuously without separation procedure. Microbeads containing a fluorophor are covalently linked to antibody or receptor. When a radiolabeled antigen or ligand is added it binds to the beads and the emitted short range electrons, excite the fluorophor in the beads. The light emitted can be measured in a scintillation counter. $^3H$ or $^{125}I$ has been used for SPA. The sensitivities achieved with SPA are comparable to the sensitivities of other procedures. SPA is applicable to immunology, receptor binding, monitoring interactions of biomolecules and study for the kinetics of interaction between receptors and ligands.

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A Study on the Durability and Synthesis of Sulfa Agents Using Glutaraldehyde (Glutaraldehyde를 이용한 설파제의 합성과 지속성에 관한 연구)

  • Yoon Cheol Hun
    • Journal of Environmental Health Sciences
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    • v.30 no.5 s.81
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    • pp.374-377
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    • 2004
  • Dual actions are the most recently used delivery system in drug study. Dual-action agents are unique chemical entities comprised of two different type of antibacterial compounds covalently linked together in a single molecule in such a way that both components are able to exert their bactericidal properties. Crosslinked sulfadiazine-sulfanilamide such as antibiotics is synthesized by synthetic handle with glutaraldehyde. As a result, New synthetic antibacterial agent exhibited the broad antibacterial activities against Gram(+) and Gram(-) of 4 strains and a long durability supposing that the stomach and blood.

Backbone Assignment of Phosphorylated Cytoplasmic Domain B of Mannitol Transporter IIMtl in Thermoanaerobacter Tengcongensis

  • Lee, Ko On;Suh, Jeong-Yong
    • Journal of the Korean Magnetic Resonance Society
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    • v.21 no.1
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    • pp.20-25
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    • 2017
  • The cytoplasmic domains A and B of the mannitol transporter enzyme $II^{Mtl}$ are covalently linked in Escherichia coli, but separately expressed in Thermoanaerobacter Tengcongensis. The phosphorylation of domain B ($TtIIB^{Mtl}$) substantially increases the binding affinity to the domain A ($TtIIA^{Mtl}$) in T. Tengcongensis. To understand the structural basis of the enhanced domain-domain interaction by protein phosphorylation, we obtained NMR backbone assignments of the phospho-$TtIIB^{Mtl}$ using a standard suite of triple resonance experiments. Our results will be useful to monitor chemical shift changes at the active site of phosphorylation and the binding interfaces.

Overexpression and Purification of Reverse Transcriptase of Retron EC83 by Changing the Downstream Sequence of the Initiation Codon

  • JEONG , DAE-WON;LIM, DONG-BIN
    • Journal of Microbiology and Biotechnology
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    • v.14 no.6
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    • pp.1280-1285
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    • 2004
  • Retron is a prokaryotic genetic element, producing a short single-stranded DNA covalently linked to RNA (msDNA-RNA) by a reverse transcriptase (RT). In retron EC83, msDNA is further processed at between the 4th and the $5^{th}$ nucleotides, leaving a 79 nucleotide-long single-stranded DNA as a final product. To investigate this site-specific cleavage in msDNA synthesis, we purified the RT protein of retron EC83. Initially, RT ORF was cloned under the tac promoter, but the expression was very poor largely because of poor translation. In order to facilitate translation, the nucleotide sequence for the first nine amino acids was randomized with synonymous codons. This change of downstream sequence of translational initiation codon greatly affected the efficiency of translation. We could isolate clones which greatly increased RT production, and their sequences were compared to those of the low producers. The overproduced protein was purified and was shown to have RT activity.

Protein Engineering of an Artificial Intersubunit Disulfide Bond Linkage in Human Dihydrolipoamide Dehydrogenase

  • Kim, Hak-Jung
    • BMB Reports
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    • v.32 no.1
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    • pp.76-81
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    • 1999
  • Dihydrolipoamide dehydrogenase (E3) belongs to the protein family of pyridine nucleotide-disulfide oxidoreductases, including glutathione reductase (GR). The two subunits of human GR are covalently linked by an intersubunit disulfide bond between the pair of the Cys-90 residues. The corresponding residue (Ser-79) in human E3 was substituted to Cys using site-directed mutagenesis. The mutant was expressed in Escherichia coli and highly purified using an affinity column. About 40% of the mutants formed a spontaneous intersubunit disulfide bond linkage. This result implies that Ser-79 and possibly surrounding residues constitute one of the several intersubunit contact regions in human E3. It provides another good piece of evidence for the predicted high degree of the structural homology between human E3 and GR. Spectroscopic studies indicate conformational changes in the mutant.

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Compiling Multicopy Single-Stranded DNA Sequences from Bacterial Genome Sequences

  • Yoo, Wonseok;Lim, Dongbin;Kim, Sangsoo
    • Genomics & Informatics
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    • v.14 no.1
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    • pp.29-33
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    • 2016
  • A retron is a bacterial retroelement that encodes an RNA gene and a reverse transcriptase (RT). The former, once transcribed, works as a template primer for reverse transcription by the latter. The resulting DNA is covalently linked to the upstream part of the RNA; this chimera is called multicopy single-stranded DNA (msDNA), which is extrachromosomal DNA found in many bacterial species. Based on the conserved features in the eight known msDNA sequences, we developed a detection method and applied it to scan National Center for Biotechnology Information (NCBI) RefSeq bacterial genome sequences. Among 16,844 bacterial sequences possessing a retron-type RT domain, we identified 48 unique types of msDNA. Currently, the biological role of msDNA is not well understood. Our work will be a useful tool in studying the distribution, evolution, and physiological role of msDNA.

Antibiotics and Durability by Action of Sulfa Agents (Sulfa제의 Dual Action에 의한 지속성과 항균성)

  • Kong, Seung-Dae;Hwang, Sung-Kwy;Yoon, Cheol-Hun;Kim, Jin-Yeong;Lee, Han-Seob
    • Journal of the Korean Applied Science and Technology
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    • v.17 no.4
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    • pp.267-272
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    • 2000
  • Dual-actions are the most recently used delivery system in drug study. Dual-action agents are unique chemical entities comprised of two different type of antibacterial compounds covalently linked together in a single molecule in such a way that both components are able to exert their bactericidal properties. Crosslinked sulfadiazine-sulfanilamide such as antibiotics is synthesized by synthetic handle with glutaraldehyde. As a result, New synthetic antibacterial agent exhibited the broad antibacterial activities against gram(+) and gram(-) of 4 strains and a long durability supposing that the stomach and blood.

Plant Light Signaling Mediated by Phytochromes and Plant Biotechnology

  • Song, Pill-Soon
    • Proceedings of the Botanical Society of Korea Conference
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    • 1998.07a
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    • pp.83-96
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    • 1998
  • The plant pigment proteins phytochromes are a molecular light sensor or switch for photomorphogenesis involving a variety of growth and developmental responses of plants to red and far-red wavelength light. Underscoring the photomorphogenesis mediated by phytochromes is the light signal transduction at molecular and cellular levels. For example, a number of genes activated by the phytochrome-mediated signal transduction cascade have been identified and characterized, especially in Arabidopsis thaliana. The light sensor/switch function of phytochromes are based on photochromism of the covalently linked tetrapyrrole chromophore between the two photoreversible forms, Pr and Pfr. The photochromism of phytochromes involves photoisomerization of the tetrapyrrole chromophore. The "photosensor" Pr-form ("switch off" conformation) of phytochromes strongly absorbs 660 nm red light, whereas the "switch on" Pfr-conformation preferentially absorbs 730 nm far-red light. The latter is generally considered to be responsible for eliciting transduction cascades of the red light signal for various responses of plants to red light including positive or negative expression of light-responsive genes in plant nuclei and chloroplasts. In this paper, we discuss the structure-function of phytochromes in plant growth and development, with a few examples of biotechnological implications.

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Design, Synthesis and Anticonvulsive Activities of Potential Prodrugs Linked by Two-carbon Chain

  • Zhao, Long-Xuan;Moon, Yoon-Soo;Basnet, Ar-Jun;Park, Jae-Gyu;Kim, Eun-kyung;Kim, Dae-Ok;Jeong, Tae-Cheon;Jahng, Yurng-Dong;Choi, Jong-Won;Lee, Eung-Seok
    • Archives of Pharmacal Research
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    • v.26 no.10
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    • pp.785-795
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    • 2003
  • For the development of new anticonvulsive agents, GABAmimetics such as nipecotic acid, isonipecotic acid, ${\gamma}-aminobutyric$ acid (GABA), ${\gamma}-vinyl$ GABA (vigabatrin) and valproic acid were covalently coupled through an ester bond by a two-carbon linker chain as potential prodrugs and evaluated for their anticonvulsive activities.