• BMB Reports
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• v.57 no.3
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• pp.135-142
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• 2024

DNA methylation is one of the most extensively studied epigenetic regulatory mechanisms, known to play crucial roles in various organisms. It has been implicated in the regulation of gene expression and chromatin changes, ranging from global alterations during cell state transitions to locus-specific modifications. 5-hydroxymethylcytosine (5hmC) is produced by a major oxidation, from 5-methylcytosine (5mC), catalyzed by the ten-eleven translocation (TET) enzymes, and is gradually being recognized for its significant role in genome regulation. With the development of state-of-the-art experimental techniques, it has become possible to detect and distinguish 5mC and 5hmC at base resolution. Various techniques have evolved, encompassing chemical and enzymatic approaches, as well as third-generation sequencing techniques. These advancements have paved the way for a thorough exploration of the role of 5hmC across a diverse array of cell types, from embryonic stem cells (ESCs) to various differentiated cells. This review aims to comprehensively report on recent techniques and discuss the emerging roles of 5hmC.

• Microbiology and Biotechnology Letters
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• v.13 no.4
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• pp.361-366
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• 1985

Distribution and substrate specificity of 5-fluorocytosine deaminase were studied in various genera of bacteria. 5-Fluorocytosine deaminase was produced by various bacteria independent of genus and species and it catalyzed the deamination of cytosine, 5-fluorocytosine and 5-methylcytosine. Xanthomonas campestris IAM 1671 produced relatively large amount of 5-fluorocytosine deaminase. The composition of optimum culture medium for enzyme production wat glycerine 0.5％, peptone 1％, yeast extract 0.5％, NaCl 0.5％ and the initial pH of the medium was 7.5. The highest enzyme formation was observed after 24 hours of cultivation In 500$m\ell$ shaking flask containing 90$m\ell$ of medium at 3$0^{\circ}C$ on a reciprocal shaker.

• Korean Journal of Microbiology
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• v.26 no.4
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• pp.368-374
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• 1988

Enzymological proprties of an extracellular cytosine deaminase from Bacellus polymyxa YL 38-3 were investigated. The extracellular enzyme was very stable, and optimum pH and temperature for the enzyme activity were found to be near pH 6.0 in 0.2M potassium phosphate buffer and at $30^{\circ}C$, respectively. 5-Fluorocytosine was converyed to 5-fluorouracil by the enzyme, but 5-methylcytosine was not to thymine by it. The enzyme activity was completely inhibited by some heavy metal ion such as 1mM of $Cd^{2-}$ and $Hg^{2+}$, and by 1mM of p-chloromercuribenzoate, respectively. The enzyme activity was inactivated about 75% by 1mM of o-phenanthroline and monoiodoacetate. But the enzyme activity was stimulated up to 200% by 1mM of 2-mercaptoethanol.

• Asian-Australasian Journal of Animal Sciences
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• v.30 no.7
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• pp.944-949
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• 2017

Objective: Investigated the effect and mechanism of ascorbic acid on the development of porcine embryos produced by somatic cell nuclear transfer (SCNT). Methods: Porcine embryos were produced by SCNT and cultured in the presence or absence of ascorbic acid. Ten-eleven translocation 3 (TET3) in oocytes was knocked down by siRNA injection. After ascorbic acid treatment, reprogramming genes were analyzed by realtime reverse transcription-polymerase chain reaction (RT-PCR). Furthermore, relative 5-methylcytosine and 5-hydroxymethylcytosine content in pronucleus were detected by realtime PCR. Results: Ascorbic acid significantly increased the development of porcine embryos produced by SCNT. After SCNT, transcript levels of reprogramming genes, Pou5f1, Sox2, and Klf were significantly increased in blastocysts. Furthermore, ascorbic acid reduced 5-methylcytosine content in pronuclear embryos compared with the control group. Knock down of TET3 in porcine oocytes significantly prevents the demethylation of somatic cell nucleus after SCNT, even if in the presence of ascorbic acid. Conclusion: Ascorbic acid enhanced the development of porcine SCNT embryos via the increased TET3 mediated demethylation of somatic nucleus.

• Journal of Microbiology and Biotechnology
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• v.14 no.6
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• pp.1182-1189
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• 2004

Cytosine deaminase (cytosine aminohydrolase, EC 3.5.4.1) stoichiometrically catalyzes the hydrolytic deamination of cytosine and 5-fluorocytosine to uracil and 5-fluorouracil, respectively. The intracellular cytosine deaminase from Chromobacterium violaceum YK 391 was purified to apparent homogeneity with 272.9-fold purification with an overall yield of $13.8\%$. The enzyme consisted of dimeric polypeptides of 63 kDa, and the total molecular mass was calculated to be approximately 126 kDa. Besides cytosine, the enzyme deaminated 5-fluorocytosine, cytidine, 6-azacytosine, and 5-methylcytosine, but not 5-azacytosine. Optimum pH and temperature for the enzyme reaction were 7.5 and $30^{\circ}C$, respectively. The enzyme was stable at pH 6.0 to 8.0, and at 30T for a week. About $70\%$ of the enzyme activity was retained at $60^{\circ}C$ for 5 min. The apparent $K_{m}$ values for cytosine, 5-fluorocytosine, and 5-methylcytosine were calculated to be 0.38 mM, 0.87 mM, and 2.32 mM, respectively. The enzyme activity was strongly inhibited by 1 mM $Hg^{2+},\;Zn^{2+},\;Cu^{2+},\;Pb^{2+},\;and\;Fe^{3+}$, and by o-phenanthroline, $\alpha,\;{\alpha}'$-dipyridyl, p-choromercuribenzoate, N-bromosuccinimide, and cWoramine­T. In addition, the enzyme activity was strongly inhibited by I mM 2-thiouracil, and weakly inhibited by 2-thiocytosine, or 5-azacytosine. Finally, intracellular and extracellular cytosine deaminases from Chromobacterium violaceum YK 391 were found to have a different optimum temperature, apparent $K_{m}$ value, and molecular mass.

• Korean Journal of Microbiology
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• v.30 no.4
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• pp.305-309
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• 1992

Cytosine deaminase (EC 3.5.4.1) from BaciNus stc~urorhermophilus was partially purified 7.2-fold with an overall yield of 52.7%. The partially purified enzyme deiiminated cytosine only.but not 5-methylcytosine and 5-fluorocytosine. The apparent Michaclis constant. Km valuefor cytosine was 5.9 mM. The enzyme was relatively stable in the range of pH 4.0 to 7.0.furthermore extremely thermo-stable : more than 75'X) of the activity was remained afterheating at 80$^{\circ}$C for I0 min at pH 6.5. The enzyme had a pH optimum at around pH7.0 to 7.5. and temperature optimum at 35 to 31$^{\circ}$C. And the activation energ (En value)determined from an Arrhenius plot was 26 Kcal/mol. The enzyme activity was stronglyinhibited by heavy metal ions such as Cd", Hg". Cut' at 1 mM, anJ by o-phenanthroline,and p-chloromcrcuribcnzoate at I mM. But the enrymc activity was activatetl increased byGMP, and CMP at 1 mM.ased by GMP, and CMP at 1 mM.

• Journal of Microbiology and Biotechnology
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• v.31 no.10
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• pp.1331-1342
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• 2021

In this study, we evaluated the mechanism of long non-coding RNA MIR22 host gene (LncRNA MIR22HG) in osteosarcoma cells. Forty-eight paired osteosarcoma and adjacent tissues samples were collected and the bioinformatic analyses were performed. Target genes and potential binding sites of MIR22HG, microRNA (miR)-629-5p and tet methylcytosine dioxygenase 3 (TET3) were predicted by Starbase and TargetScan V7.2 and confirmed by dual-luciferase reporter assay. Cell Counting Kit-8, colony formation and flow cytometry assays were utilized to determine the viability, proliferation and apoptosis of transfected osteosarcoma cells. Pearson's analysis was introduced for the correlation analysis between MIR22HG and miR-629-5p in osteosarcoma tissue. Relative expressions of MIR22HG, miR-629-5p and TET3 were measured by quantitative real-time polymerase chain reaction or Western blot. MiR-629-5p could competitively bind with and was negatively correlated with MIR22HG, the latter of which was evidenced by the high expression of miR-629-5p and low expression of MIR22HG in osteosarcoma tissues. Overexpressed MIR22HG repressed the viability and proliferation but enhanced apoptosis of osteosarcoma cells, which was reversed by miR-629-5p upregulation. TET3 was the target gene of miR-629-5p, and the promotive effects of upregulated miR-629-5p on the viability and proliferation as well as its repressive effect on apoptosis were abrogated via overexpressed TET3. To sum up, overexpressed MIR22HG inhibits the viability and proliferation of osteosarcoma cells, which was achieved via regulation of the miR-629-5p/TET3 axis.

• Journal of Microbiology and Biotechnology
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• v.9 no.2
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• pp.173-178
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• 1999

The extracellular cytosine deaminase (EC 3.5.4.1) from Chromobacterium violaceum YK 391 was purified 264.7-fold with an overall yield of 14.3%. The enzyme was for the first time homogeneous by the criteria of polyacrylamide gel electrophoresis performed in the absence and in the presence of sodium dodecyl sulfate. The molecular weight of the purified enzyme was estimated to be about 156 kDa. The enzyme consisted of two identical subunits of approximate molecular weight 78 kDa. The isoelectric point of the enzyme was pH 5.55. The enzyme had a pH optimum of 7.5 and a temperature optimum of around 40 to $45^{\circ}C$. Besides cytosine, the enzyme deaminated 5-fluorocytosine, cytidine, 5-methylcytosine, and 6-azacytosine, but not 5-azacytosine. The extracellular cytosine deaminase is believed to be unique because it was active not only on cytosine but also on cytidine. The apparent $K_m$ values for cytosine, 5-fluorocytosine, cytidine, and 5-methylcytosine were determined to be 1.55 mM, 5.52 mM, 10.4 mM, and 67.2 mM, respectively. The enzyme activity was strongly inhibited by heavy metal ions such as $Fe^{2+},Pb^{2+},Cd^{2+},Zn^{2+}, Hg^{2+}, and Cu^{2+}$ at 1 mM, and completely by $\alpha,\alpha$'-dipyridyl, and $\rho$-chloromercuribenzoate at 1 mM, and weakly inhibited by 1mM ο-phenanthroline. The enzyme activity was not affected by various nucleosides and nucleotides.

• Proceedings of the Korean Society of Developmental Biology Conference
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• 2003.10a
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• pp.26-28
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• 2003

"Epigenetics" means the study of heritable changes in gene-activity without changes in DNA sequences. Methylation of the cytosine residue in a CpG dinucleotide sequence is a characteristic of the vertebrate genome. In vertebrates, methylation of DNA mainly occurs at the 5′-position of cytosine in a CpG dinucleotide forming 5-methylcytosine. Methylation of DNA plays a profound role in transcriptional repression of gene expression through several mechanisms. Generally, DNA of inactive genes is more heavily methylated than that of active ones; conversely demethylation of DNA reactivates gene expression in vivo and in vitro.

• Molecules and Cells
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• v.38 no.11
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• pp.925-935
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• 2015

DNA methylation is a well-characterized epigenetic modification that plays central roles in mammalian development, genomic imprinting, X-chromosome inactivation and silencing of retrotransposon elements. Aberrant DNA methylation pattern is a characteristic feature of cancers and associated with abnormal expression of oncogenes, tumor suppressor genes or repair genes. Ten-eleven-translocation (TET) proteins are recently characterized dioxygenases that catalyze progressive oxidation of 5-methylcytosine to produce 5-hydroxymethylcytosine and further oxidized derivatives. These oxidized methylcytosines not only potentiate DNA demethylation but also behave as independent epigenetic modifications per se. The expression or activity of TET proteins and DNA hydroxymethylation are highly dysregulated in a wide range of cancers including hematologic and non-hematologic malignancies, and accumulating evidence points TET proteins as a novel tumor suppressor in cancers. Here we review DNA demethylation-dependent and -independent functions of TET proteins. We also describe diverse TET loss-of-function mutations that are recurrently found in myeloid and lymphoid malignancies and their potential roles in hematopoietic transformation. We discuss consequences of the deficiency of individual Tet genes and potential compensation between different Tet members in mice. Possible mechanisms underlying facilitated oncogenic transformation of TET-deficient hematopoietic cells are also described. Lastly, we address non-mutational mechanisms that lead to suppression or inactivation of TET proteins in cancers. Strategies to restore normal 5mC oxidation status in cancers by targeting TET proteins may provide new avenues to expedite the development of promising anti-cancer agents.