• Korean Journal of Medicinal Crop Science
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• v.15 no.5
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• pp.339-345
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• 2007

Field performance and morphological characterization was conducted on seven transgenic lines of Codonopsis lanceolata expressing ${\gamma}-TMT$ gene. The shoots were obtained from leaf explants after co-cultivation with Agrobacterium tume-faciens strain LBA 4404 harboring a binary vector pYBI 121 that carried genes encoding ${\gamma}-Tocopherol$ methyltransferase gene (${\gamma}-TMT$) and a neomycin phosphotransferase II gene (npt II) for kanamycin resistance. The transgenic plants were transferred to a green house for acclimation. Integration of T-DNA into the $T_0\;and\;T_1$ generation of transgenic Codonopsis lanceolata genome was confirmed by the polymerase chain reaction and southern blot analysis. The progenies of transgenic plants showed phenotypic differences within the different lines and with relative to control plants. When grown in field, the transgenic plants in general exhibited increased fertility, significant improvement in the shoot weight, root weight, shoot height and rachis length with relation to the control plants. However, all seven independently derived transgenic lines produced normal flower with respect to its shape, size, color and seeds number at its maturity. Indicating that the addition of a selectable marker gene in the plant genome does not effect on seed germination and agronomic performance of transgenic Codonopsis lanceolata. $T_1$ progenies of these plants were obtained and evaluated together with control plant in a field experiment. Overall, the agronomic performance of $T_1$ progenies of transgenic Codonopsis lanceolata showed superior to that of the seed derived non-transgenic plant. In this study, we report on the morphological variation and agronomic performance of transgenic Codonopsis lanceolata developed by Agrobacterium transformation.

• Korean Journal of Microbiology
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• v.48 no.2
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• pp.79-86
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• 2012

ErmSF is one of the Erm family proteins which catalyze S-adenosyl-$_L$-methionine dependent modification of a specific adenine residue (A2058, E. coli numbering) in bacterial 23S rRNA, thereby conferring resistance to clinically important macrolide, lincosamide and streptogramin B ($MLS_B$) antibiotics. $^{222}FXPXPXVXS^{230}$ (ErmSF numbering) sequence appears to be a consensus sequence among the Erm family. This sequence was supposed to be involved in direct interaction with the target adenine from the structural studies of Erm protein ErmC'. But in DNA methyltarnsferase M. Taq I, this interaction have been identified biochemically and from the complex structure with substrate. Arginine 223 and 227 in this sequence are not conserved among Erm proteins, but because of the basic nature of residues, it was expected to interact with RNA substrates. Two amino acid residues were replaced with Ala by site-directed mutagenesis. Two mutant proteins still maintained its activity in vivo and resistant to the antibiotic erythromycin. Compared to the wild-type ErmSF, R223A and R227A proteins retained about 50% and 88% of activity in vitro, respectively. Even though those arginine residues are not essential in the catalytic step, with their positive charge they may play an important role for RNA binding.

• Korean Journal of Microbiology
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• v.52 no.4
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• pp.487-494
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• 2016

In Saccharomyces cerevisiae, Paf1 complex consists of five proteins, and they are structurally and functionally well conserved in yeast, fruit fly, plants, and human. With binding to RNA polymerase II from transcription start site to termination site, Paf1 complex functions as a platform for recruiting many types of transcription factors to RNA polymerase II. Paf1 complex contributes to H2B ubiquitination and indirectly influences on H3K4 di- and tri-methylation by histone crosstalk. But the individual effects of five components in Paf1 complex on these two histone modifications including H2B ubiquitination and H3K4 methylation largely remained to be identified. In this study, we constructed the single-gene knockout mutants of each Paf1 complex component and observed H3K4 mono-, di-, and trimethylation as well as H2B ubiquitination in these mutants. Interestingly, in each ${\Delta}paf1$, ${\Delta}rtf1$, and ${\Delta}ctr9$ strain, we observed the dramatic defect in H3K4 monomethylation, which is independent of H2B ubiquitination, as well as H3K4 di- and trimethylation. However, the protein level of Set1, which is methyltransferase for H3K4, was not changed in these mutants. This suggests that Paf1 complex may directly influence on H3K4 methylation by directly regulating the activity of Set1 or the stability of Set1 complex in an H2B ubiquitination independent manner.

• Journal of Physiology & Pathology in Korean Medicine
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• v.17 no.3
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• pp.633-642
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• 2003

A pain is the symptom which defends against noxious stimulus about a human body, it is known that if the periphery of perceptive nerve were stimulated by a physical or chemical factors, the stimulation is induced by transmission to pain center in the cerebral cortex according to pain conduction tract. The treatment of pain is to decrease a stimulus that causes a pain or block off a nerve transmitting a stimulus or puts on a way to calm down pain center, but It is for adjustment of a pain to be the most representative in acupuncture among various ways to cure a pain in Oriental medicine. However, the analgesic effect of an individual response to acupuncture stimulation shows marked individual variations, so these days genetic a few approach is attempted. On this the author determined that the responding group was appointed those whose tail flick latency (TFL) responding time delayed the minimum of 30 % comparing with basal reaction time. For those whose TFL time had shorter than 30 % was grouped as a non-responding group. And then the hypothalamus of each group was dissected and RNA was further purified. After synthesizing cDNA using oligo dT primer, products were finally applied to the PCR. The results were as follows; The ratio of responding group to non-responding group was 6:4. Ach T (acetylcholinesterase T subunit), BF-I (Brain factor-I), DBH (Dopamine β-hydroxylase) and PNM (Phosphotidylethanolamine N-Methyltransferase) were revealed significantly in the responding group. Cathepsin B and Tau were revealed significantly in the non-responding group. The PCR results show that Ach T, BF-I, DBH and PNM are expressed abundantly in the responding group, where as cathepsin B and tau are abundant in the non-responding group. These results suggest that the analgesic effect on acupuncture stimulation is related to regulation of neurotransmitter as well as neurodegeration of cerebrum.

• Journal of Plant Biotechnology
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• v.36 no.1
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• pp.23-29
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• 2009

We have previously isolated a CCCH type zinc-finger protein gene, OsZF2 (Oryza sativa Zinc Finger 2), from the cold-treated rice cDNA library. To investigate the potential role of OsZF2, transgenic rice lines over-expressing OsZF2 under the control of CaMV 35S promoter have been developed through Agrobacterium-mediated transformation. Elevated level of OsZF2 transcripts was confirmed by RNA gel blot analysis in transgenic rice. Under the 100 mM NaCl condition, the transgenic rice showed significantly enhanced growth rate in terms of shoot length and fresh weight, implicating that OsZF2 is likely to be involved in salt response of rice. In the field condition, however, the transgenic rice showed a dwarf phenotype and flowering time was delayed. Genome expression profiling analysis of transgenic plants using the 20K NSF rice oligonucleotide array revealed many up-regulated genes related to stress responses and signaling pathways such as chaperone protein dnaJ 72, salt stress-induced protein, PR protein, disease resistance proteins RPM1 and Cf2/Cf5 disease resistance protein, carbohydrate/ sugar transporter, OsWAK kinase, brassinosteroid LRR receptor kinase, and jasmonate O-methyltransferase. These data suggest that the CCCH type zinc-finger protein OsZF2 is a upstream transcriptional factor regulating growth and stress responsiveness of rice.

• Asian Pacific Journal of Cancer Prevention
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• v.14 no.11
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• pp.6261-6265
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• 2013

Background: Recent studies have suggested that expression of the RAS protein activator like-1 gene (RASAL1) is decreased in gastric carcinoma tissues and cell lines, indicated a role in tumorigenesis and development of gastric cancer. Reduced expression of RASAL1 could result in aberrant increase of activity of RAS signaling pathways in cancer cells. However, the exact mechanism which induces down-regulation of the RASAL1 gene remains unclear. This study aimed to determine the methylation status and regulation of RASAL1 in gastric cancer. Materials and Methods: Using the methylation-specific polymerase chain reaction (MSP), the methylation status of CpG islands in the RASAL1 promoter in gastric cancers and paired adjacent non-cancerous tissues from 40 patients was assessed and its clinicopathological significance was analyzed. The methylation status of RASAL1 in gastric cancer lines MKN-28, SGC-790l, BGC-823, as well as in normal gastric epithelial cell line GES-l was also determined after treatment with a DNA methyltransferase inhibitor, 5-aza-2'-doexycytidine (5-Aza-CdR). RAS activity (GAS-GTP) was assessed through a pull-down method, while protein levels of ERK1/2, a downstream molecule of RAS signaling pathways, were determined by Western blotting. Results: The frequencies of RASAL1 promoter methylation in gastric cancer and paired adjacent non-cancerous tissues were 70% (28/40) and 30% (12/40) respectively (P<0.05). There were significantly correlations between RASAL1 promoter methylation with tumor differentiation, tumor size, invasive depth and lymph node metastasis in patients with gastric cancer (all P<0.05), but no correlation was found for age or gender. Promoter hypermethylation of the RASAL1 gene was detected in MKN-28, SGC-790l and BGC-823 cancer cells, but not in the normal gastric epithelial cell line GES-1. Elevated expression of the RASAL1 protein, a decreased RAS-GTP and p-ERK1/2 protein were detected in three gastric cancer cell lines after treatment with 5-Aza-CdR. Conclusions: Aberrant hypermethylation of the RASAL1 gene promoter frequently occurs in gastric cancer tissues and cells. In addition, the demethylating agent 5-Aza-CdR can reverse the hypermethylation of RASAL1 gene and up-regulate the expression of RASAL1 significantly in gastric cancer cells in vivo. Our study suggests that RASAL1 promoter methylation may have a certain relationship with the reduced RASAL1 expression in gastric cancer.

• Asian Pacific Journal of Cancer Prevention
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• v.17 no.4
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• pp.2185-2193
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• 2016

Background: The pathogenesis of sporadic colorectal cancer (CRC) is influenced by the patient genetic background and environmental factors. Based on prior understanding, these are classified in two major pathways of genetic instability. Microsatellite instability (MSI) and CPG island methylator phenotype (CIMP) are categorized as features of the hypermethylated prototype, and chromosomal instability (CIN) is known to be indicative of the non-hypermethylated category. Secreted frizzled related protein 2 (SFRP2), APC1A in WNT signaling pathway and the DNA repair gene, O6-methylguanine-DNA methyltransferase (MGMT), are frequently hypermethylated in colorectal cancer. Detection of methylated DNA as a biomarker by easy and inexpensive methods might improve the quality of life of patients with CRC via early detection of cancer or a precancerous condition. Aim: To evaluate the rate of SFRP2 and MGMT hypermethylation in both polyp tissue and serum of patients in south Iran as compared with matched control normal population corresponding samples. Materials and Methods: Methylation-specific PCR was used to detect hypermethylation in DNA extracted from 48 polypoid tissue samples and 25 healthy individuals. Results: Of total polyp samples, 89.5% had at least one promoter gene hypermethylation. The most frequent methylated locus was SFRP2 followed by MGMT-B (81.2 and 66.6 percent respectively). Serologic detection of hypermethylation was 95% sensitive as compared with polyp tissue. No hypermethylation was detected in normal tissue and serum and its detection in patients with polyps, especially of serrated type, was specific. Conclusions: Serologic investigation for detection of MGMT-B, SFRP2 hypermethylation could facilitate prioritization of high risk patients for colonoscopic polyp detection and excision.

• Asian-Australasian Journal of Animal Sciences
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• v.31 no.4
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• pp.595-606
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• 2018

Objective: The Fusarium mycotoxins of deoxynivalenol (DON) and zerolenone (ZEN) cause health hazards for both humans and farm animals. Therefore, the main intention of this study was to reveal DON and ZEN effects on the mRNA expression of pro-inflammatory cytokines and other immune related genes in the liver of piglets. Methods: In the present study, 15 six-week-old piglets were randomly assigned to the following three different dietary treatments for 4 weeks: control diet, diet containing 8 mg DON/kg feed, and diet containing 0.8 mg ZEN/kg feed. After 4 weeks, liver samples were collected and sequenced using RNA-Seq to investigate the effects of the mycotoxins on genes and gene networks associated with the immune systems of the piglets. Results: Our analysis identified a total of 249 differentially expressed genes (DEGs), which included 99 upregulated and 150 downregulated genes in both the DON and ZEN dietary treatment groups. After biological pathway analysis, the DEGs were determined to be significantly enriched in gene ontology terms associated with many biological pathways, including immune response and cellular and metabolic processes. Consistent with inflammatory stimulation due to the mycotoxin-contaminated diet, the following Kyoto encyclopedia of genes and genomes pathways, which were related to disease and immune responses, were found to be enriched in the DEGs: allograft rejection pathway, cell adhesion molecules, graft-versus-host disease, autoimmune thyroid disease (AITD), type I diabetes mellitus, human T-cell leukemia lymphoma virus infection, and viral carcinogenesis. Genome-wide expression analysis revealed that DON and ZEN treatments downregulated the expression of the majority of the DEGs that were associated with inflammatory cytokines (interleukin 10 receptor, beta, chemokine [C-X-C motif] ligand 9), proliferation (insulin-like growth factor 1, major facilitator superfamily domain containing 2A, insulin-like growth factor binding protein 2, lipase G, and salt inducible kinase 1), and other immune response networks (paired immunoglobulin-like type 2 receptor beta, Src-like-adaptor-1 [SLA1], SLA3, SLA5, SLA7, claudin 4, nicotinamide N-methyltransferase, thyrotropin-releasing hormone degrading enzyme, ubiquitin D, histone $H_2B$ type 1, and serum amyloid A). Conclusion: In summary, our results demonstrated that high concentrations DON and ZEN disrupt immune-related processes in the liver.

• Biomedical Science Letters
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• v.11 no.2
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• pp.165-173
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• 2005

A potent demethylating agent, 5-Azacytidine (5-AzaC) has been widely used as in many studies on DNA methylation, regulation of gene expression, and cancer biology. The mechanisms of the demethylating activity were known to be formation of complex between DNA and DNA methyltransferase (MTase), which depletes cellular MTase activity. However, 5-AzaC can also induce hypermethylation of a transgene in a transgenic cell line, G12 cells and it was explained as a result of defense mechanisms to inactivate foreign gene(s) somehow. This finding evoked the question that whether the phenomenon of hypermethylation induced by 5-AzaC is limited to the transgene or it can be occurred in endogenous gene(s). In order to answer the question, mutagenicity test of 5-AzaC and molecular characterization of mutants obtained from the test were performed using an endogenous gene, thymidine kinase (tk) in Chinese hamster V79 cells. When V79 and V79-J3 subclone cells were treated with 1, 2.5 ,5, $10{\mu}M$ of 5-AzaC for 48 hours, their maximum mutant frequencies were revealed as $6\times10^{-3}\;at\;5{\mu}M$(350-fold induction over background) and $8\times10^{-3}\;at\;2.5{\mu}M$ (l,800-fold induction over background) respectively. Since the induction rates were too high to be induced by true mutations, many trifluorothymidine (TFT)-resistant $(TFT^R)$ cells were subjected to Northern blot analysis to check the presence of tk transcripts. Surprisingly, all clones tested possessed the transcripts in a similar level, that implicates the $TFT^R$ phenotype induced by 5-AzaC has not given rise to hypermethylation of the gene in spite of unusually high mutation frequency. In addition, it has shown that the TK activity in the pool of 5-AzaC-induced $TFT^R$ cells has about a half of that in spontaneously-induced $TFT^R$ cells or in non-selected parental V79-J3 cells. This result suggests that the mechanism(s) underlying the TFT-resistance between spontaneously occurred and 5-AzaC-induced cells may be different. These findings have shown that the $TFT^R$ phenotype induced by 5-AzaC has not given rise to hypermethylation of the tk gene, and 5-AzaC may be induced by one or combined pathways among many drug resistance mechanisms. The exact mechanisms for the 5-AzaC-induced $TFT^R$ phenotype remain to elucidate.

• Journal of Life Science
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• v.27 no.4
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• pp.398-407
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• 2017

BTG 1 (B-cell translocation gene 1) gene was first identified as a translocation gene in a case of B-cell chronic lympocytic leukemia. BTG1 is a member of the BTG/TOB family with sharing a conserved N-terminal region, which shows anti-proliferation properties and is able to stimulate cell differentiation. In this study, we identified and characterized the pacific oyster Crassostrea gigas BTG1 (cg-BTG1) gene from the gill cDNA library by an Expressed Sequence Tag (EST) analysis and its nucleotide sequence was determined. The cg-BTG1 gene encodes a predicted protein of 182 amino acids with 57% 56% identities to its zebrafish and human counterparts, and is an intron-less gene, which was confirmed by PCR analysis of genomic DNA. Maximal homologies were shown in conserved Box A and B. The deduced amino acid sequence shares high identity with other BTG1 genes of human, rat, mouse and zebrafish. The phylogenic analysis and sequence comparison of cg-BTG1 with other BTG1 were found to be closely related to the BTG1 gene structure. In addition, the predicted promoter region and the different transcription-factor binding site like an activator protein-1 (AP-1) response element involved in negative regulation and serum response element (SRE) were able to be identified by the genomic DNA walking experiment. The quantitative real-time PCR analysis showed that the mRNA of cg-BTG1 gene was expressed in gill, heart, digestive gland, intestine, stomach and mantle. The cg-BTG1 gene was expressed mainly in heart and mantle.