• Journal of Plant Biology
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• v.32 no.4
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• pp.331-341
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• 1989

DNA methyltransferase was purified 282.6-fold from Chlamydomonas reinhardtii 21gr (mt+) gametic cell to examine the effect of polyamine on the enzyme acctivity. Polyacrylamide gel electrophoresis(PAGE) revealed at least three bands(1 major band, 2 minor bands). Among these, the major band represents DNA methyltransferase. Polyacrylamide gel electrophoresis in the presence of 0.1% sodium dodecylsulfate(SDS-PAGE) revealed a major band with M.W. 60,000. DNA methyltransferase activity was inhibited more effectively by spermine than by spermidine, and the inhibition by putrescine was smaller than spermine and spermidine. DNA methyltransferase activity was inhibited by 40% and 53% at 5mM and 20mM spermine, respectively. In the case of spermidine, the inhibition was 35% at 10mM and 44% at 20mM. However, the inhibition by putrescine appeared only above 5mM and reached about 25% at 20mM.

• Applied Biological Chemistry
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• v.45 no.4
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• pp.203-206
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• 2002

${\gamma}-Tocopherol$ methyltransferase (TMT) is an enzyme catalyzing ${\gamma}-tocopherol$ into ${\alpha}-tocopherol$ at the final step of ${\alpha}-tocopherol$ synthesis pathway. Putative TMT cDNA clone specific to Perilla frutescens immature seeds was isolated from cDNA library. The cDNA clone consisted of 1369 bp open reading frame encoding 369 amino acids with a relative Mw of 42 kDa. Results revealed the CDNA has 60% homology to Arabidopsis thaliana TMT, and possesses methyltransferase and S-adenosyl methionine-binding domains, suggesting that cDNA encodes a ${\gamma}-tocopherol$ methyltransferase To characterize the properties of the TMT gene, the cDNA sequences coding for mature TMT were expressed in E. coli and assayed to determine the enzyme activity in vitro.

• BMB Reports
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• v.35 no.3
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• pp.348-351
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• 2002

AquI DNA methyltransferase, M.AquI, catalyses the transfer of a methyl group from S-adenosyl-L-methionine to the C5 position of the outermost deoxycytidine base in the DNA sequence 5'CYCGRG3'. M.AquI is encoded by two overlapping ORFs (termed $\alpha$ and $\beta$) instead of the single ORF that is customary for Class II methyltransferase genes. The structural organization of the M.AquI protein sequence is quite similar to that of other bacterial C5-DNA methyltransferases. Ten conserved motifs are also present in the correct order, but only on two polypeptides. We separately subcloned the genes that encode the $\alpha$ and $\beta$ subunits of M.AquI into expression vectors. The overexpressed His-fusion $\alpha$ and $\beta$ subunits of the enzyme were purified to homogeneity in a single step by Nickel-chelate affinity chromatography. The purified recombinant proteins were assayed for biological activity by an in vitro DNA tritium transfer assay. The $\alpha$ and $\beta$ subunits of M.AquI alone have no DNA methyltransferase activity, but when both subunits are included in the assay, an active enzyme that catalyses the transfer of the methyl group from S-adenosyl-L-methionine to DNA is reconstituted. We also showed that the $\beta$ subunit alone contains all of the information that is required to generate recognition of specific DNA duplexes in the absence of the $\alpha$ subunit.

• Korean Journal of Microbiology
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• v.55 no.2
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• pp.103-111
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• 2019

DNA methylation is involved in diverse processes in bacteria, including maintenance of genome integrity and regulation of gene expression. CcrM, the DNA methyltransferase conserved in Alphaproteobacterial species, carries out $N^6$-adenine or $N^4$-cytosine methyltransferase activities using S-adenosyl methionine as a co-substrate. Celeribacter marinus IMCC12053 from the Alphaproteobacterial group was isolated from a marine environment. Single molecule real-time sequencing method (SMRT) was used to detect the methylation patterns of C. marinus IMCC12053. Gibbs motif sampler program was used to observe the conversion of adenosine of 5'-GANTC-3' to $N^6$-methyladenosine and conversion of $N^4$-cytosine of 5'-GpC-3' to $N^4$-methylcytosine. Exocyclic DNA methyltransferase from the genome of strain IMCC12053 was chosen using phylogenetic analysis and $N^4$-cytosine methyltransferase was cloned. IPTG inducer was used to confirm the methylation activity of DNA methylase, and cloned into a pQE30 vector using dam-/dcm- E. coli as the expression host. The genomic DNA and the plasmid carrying methylase-encoding sequences were extracted and cleaved with restriction enzymes that were sensitive to methylation, to confirm the methylation activity. These methylases protected the restriction enzyme site once IPTG-induced methylases methylated the chromosome and plasmid, harboring the DNA methylase. In this study, cloned exocyclic DNA methylases were investigated for potential use as a novel type of GpC methylase for molecular biology and epigenetics.

• The Korean Journal of Mycology
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• v.21 no.4
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• pp.279-284
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• 1993

The metE gene coding for $N^{5}-methyl-H_{4}-folate;$ homocysteine methyltransferase from the basidiomycete Ganoderma lucidum was cloned by complementation of methionine-requiring mutants of E. coli. The size of a inserted DNA was about 1.54 kb and had 5 restriction enzyme sites. A physical map was constructed. Southern blot analysis confirmed the presence of a transforming DNA in the genome of Ganoderma lucidum. indicating the presence of a single copy.

• Proceedings of the Korean Society of Developmental Biology Conference
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• 2003.10a
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• pp.84-84
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• 2003

DNA methylation is involved in epigenetic processes such as X-chromosome inactivation, imprinting and silencing of transposons. DNA methylation is a highly plastic and critical component of mammalian development The DNA methyltransferases (Dnmts) are responsible for the generation of genomic methylation patterns, which lead to transcriptional silencing. The maintenance DNA methyltransferase enzyme, Dnmt 1, and the de novo methyltransferase, Dnmt3a and Dnmt3b, are indispensable for development because mice homozygous for the targeted disruption of any of these genes are not viable. The occurrence of DNA methylation is not random, and it can result in gene silencing The mechanisms underlying these processes are poorly understood. It is well established that DNA methylation and histone deacetylation operate along a common mechanistic pathway to repress transcription through the action of methyl-binding domain proteins (MBDs), which are components of, or recruit, histone deacetylase (HDAC) complexes to methylated DNA. As a basis for future studies on the role of the DNA-methyl-transferase in porcine development, we have isolated and characterized a partial cDNA coding for the porcine Dnmt1. Total RNA of testis, lung and ovary was isolated with TRlzol according to the manufacture's specifications. 5 ug of total RNA was reverse transcribed with Super Script II in the presence of porcine Dnmt 1 specific primers. Standard PCRs were performed in a total volume of 50 ul with cDNA as template. Two DNA fragmenets in different position were produced about 700bp, 1500bp and were cloned into pCR II-TOPO according to the manufacture's specification. Assembly of all sequences resulted in a cDNA from 158bp of 5'to 4861bp of 3'compare with the known human maintenance methyltransferase. Now, we are cloning the unknown Dnmt 1 region by 5'-RACE method and expression of Dnmt 1 in tissues from adult porcine animals.

• Reproductive and Developmental Biology
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• v.34 no.4
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• pp.283-288
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• 2010

During normal early embryonic development in mammals, the global pattern of genomic DNA methylation undergoes marked. changes. The level of methylation is high in male and female gametes. Thus, we cloned the cDNA of the porcine DNA methyltransferase 1 (Dnmt1) gene to promote the efficiency of the generation of porcine clones. In this study, porcine Dnmt1 cDNA was sequenced, and Dnmt1 mRNA expression was detected by reverse transcription-polymerase reaction (RT-PCR) in porcine tissues during embryonic development. The porcine Dnmt1 cDNA sequence showed more homology with that of bovine than human, mouse, and rat. The complete sequence of porcine Dnmt1 cDNA was 4,774-bp long and consisted of an open reading frame encoding a protein of 1611 amino acids. The amino acid sequence of porcine DNMT1 showed significant homology with those of bovine (91%), human (88%), rat (76%), and mouse (75%) Dnmt1. The expression of porcine Dnmt1 mRNA was detected during porcine embryogenesis. The mRNA was detected at stages of porcine preimplantation development (1-cell, 2-cell, 4-cell, 8-cell, morula, and blastocyst stages). It was also abundantly expressed in tissues (lung, ovary, kidney and somatic cells). Further investigations are necessary to understand the complex links between methyltransferase 1 and the transcriptional activity in cloned porcine tissues.

• Journal of the Korean Society of Tobacco Science
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• v.25 no.2
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• pp.87-94
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• 2003

Recently, many researches for plant alkaloids, one of the largest groups of natural products, are reported because of their various pharmacological activity. This study was carried out to clone putrescine N-methyltransferase (PMT) gene which is a key enzyme in diverting polyamine metabolism towards the biosynthesis of nicotine and related alkaloids from Burley tobacco. To induce expression of PMT gene in tobacco plant, the floral meristem was removed and then mRNA was purified from root. cDNA encoding PMT gene was isolated by RT PCR and cloned. Three different groups of clones were screened by PCR and restriction enzyme digestion analysis and were characterized. The data of these screening revealed that three types of PMT are present in Burley tobacco. Comparison of the nucleotide sequence of this three genes encoding putative PMT with those of other tobaccos revealed that two types of PMT are newly discovered from Nicotiana tabacum cv. Br21 tobacco and they were same as PMT2, PMT3 of N. tabacum cv. Xanthi.

• Journal of Plant Biology
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• v.34 no.4
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• pp.267-273
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• 1991

Polyamine levels in the male and female cells as well as DNA methyltransferase activity in the female cells during gametogenesis of Chlamydomonas reinhardtii indicated that both spermidine and spermine levels were decreased while DNA methyltransferase activity was markedly increased about 12 hours after the onset of gametogenesis. In vitro, putrescine and spermine at 1 mM inhibited methylation of chloroplasts DNA isolated from vegetative female cells by 35% and 65%, respectively. Spermine was found to be more inhibitory than putrescine at all concentrations tested. The pattern of the inhibition by polyamines appeared different from that caused by cations. The results obtained in this work suggest that the polyamine inhibition of DNA methylation is due to an action of polyamines on the enzyme involved instead of on the DNA itself.

• Journal of Life Science
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• v.18 no.4
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• pp.580-584
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• 2008

$O^6-methylguanine-DNA$ methyltransferase (MGMT) is a DNA repair protein that protects cells against the carcinogenic effects of alkylating agents. The loss of MGMT expression was commonly known due to hypermethylation of CpG islands in its promoter region. We evaluated the expression of MGMT by immunohistochemistry in order to examine the relationship between loss of MGMT expression and clinicopathological characteristics in 74 Korean patients with non-small cell lung cancers. Loss of MGMT was detected in 25 (33.8%) of 74 cases. The loss of MGMT expression was frequently seen in the adenocarcinoma than in the squamous cell carcinoma (p=0.021). However, there was no significant differences between loss of MGMT expression and other clinicopathological characteristics, including age, gender, smoking status, tumor size, tumor T stage, and lymph node metastasis (p>0.05). In conclusion, loss of MGMT expression was related with the histologic type of lung cancer. Further methylation study of MGMT promoter is needed to evaluate the relationships with immunohistochemical expression of MGMT and to clarify the role of MGMT in lung cancer.