• Journal of Plant Biotechnology
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• v.38 no.1
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• pp.87-93
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• 2011

S-Adenosylmethionine decarboxylase (SAMDC; EC 4.1.4.50), a key enzyme for polyamines biosynthesis, was tightly regulated for homeostatic levels. Carnation SAMDC gene (CSDC9) has an small upstream open reading frame (uORF) of 54 amino acids in 5'-leader sequence. To explore the functional mechanism of uORFs in controlling translation, we used a GUS reporter gene driven with the 35S promoter and uORF region of SAMDC gene for making transgenic tobacco plants. In our experiment, there were a translational inhibition of its downstream GUS ORF by SAMDC uORF sequence or SAMDC uORF protein. Expecially, translational inhibition was most effective in point-mutated construct, in which the start codon was changed. Therefore, this results suggested the ribosomal stalling might be involved in this translational inhibitory process. The frame shift in amino acid sequence of SAMDC uORF with start codon and stop codon resulted in a moderate increasing in GUS activity, suggesting the native amino acid sequence was important for a function as a translational inhibitor. Also, we showed that the production of GUS protein was significantly inhibited in the presence of the small uORF using histochemical analysis of GUS expression in seedlings and tobacco flowers. Importantly, the small uORF sequence induced a real peptide of 5.7 kDa, which was provided the presence of SAMDC uORF peptide band using an in vitro transcription/translation system. The peptide product of uORF might interact with other components of translational machinery as well as polyamines, which was resulted from that polyamine treatment was inhibited GUS protein band in SDS-PAGE experiment.

• Journal of Plant Biotechnology
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• v.36 no.4
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• pp.384-391
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• 2009

Previously, two events (H15 and B20) of transgenic pepper (Capsicum annuum L.) that enhanced resistance to Cucumber mosaic virus (CMV) by the introduction of CMV coat protein (CP) gene were constructed. Presently, a single copy number of the CP gene was revealed in H15 and B20 by Southern blot. To predict possible unintended effects due to transgene insertion in an endogenous gene, we carried out sequencing of the 5'-flanking region of the CP gene and a Blastbased search. The results revealed that insertion of the transgene into genes encoding putative proteins may occur in the H15 and B20 transgenic event. Mutiplex polymerase chain reaction (PCR) for simultaneous detection and identification of transgenic pepper was conducted with a set of nine primers. Both transgenic event were differentiated from non-transgenic event by the presence of 267 bp and 430 bp PCR products indicative of CP gene specific primer pairs and primer pairs targeting the CP gene and 35S promoter. H15 and B20 uniquely possessed a 390 bp and 596 bp PCR product, respectively. The presence of a 1115 bp product corresponding to intrinsic pepper actin gene confirmed the use of pepper DNA as the PCR template. The primer set and PCR conditions used presently may allow the accurate and simple identification of CMV resistant transgenic pepper.

• Korean Journal of Head & Neck Oncology
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• v.33 no.1
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• pp.21-29
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• 2017

Methylation of CpG islands in the promoter region of genes acts as a significant mechanism of epigenetic gene silencing in head and neck squamous cell carcinoma (HNSCC). DNA methylation markers are particularly advantageous because DNA methylation is an early event in tumorigenesis, and the epigenetic modification, 5-methylcytosine, is a stable mark. In the present study, we assessed the genome-wide preliminary screening and were to identify novel methylation biomarker candidate in HNSCC. Genome-wide methylation analysis was performed on 10 HNSCC tumors using the Methylated DNA Isolation Assay (MeDIA) CpG island microarray. Validation was done using immunohistochemistry using tissue microarray of 135 independent HNSCC tumors. In addition, in vitro proliferation, migration/invasion assays, RT-PCR and immunoblotting were performed to elucidate molecular regulating mechanisms. Our preliminary validation using CpG microarray data set, immunohisto-chemistry for HNSCC tumor tissues and in vitro functional assays revealed that methylation of the Homeobox B5 (HOXB5) and H6 Family Homeobox 2 (HMX2) could be possible novel methylation biomarkers in HNSCC.

• Molecules and Cells
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• v.25 no.4
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• pp.559-565
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• 2008

Members of the TGA family of basic domain/leucine zipper transcription factors regulate defense genes through physical interaction with NON-EXPRESSOR OF PR1 (NPR1). Of the seven TGA family members, TGA4/octopine synthase (ocs)-element-binding factor 4 (OBF4) is the least understood. Here we present evidence for a novel function of OBF4 as a regulator of flowering. We identified CONSTANS (CO), a positive regulator of floral induction, as an OBF4-interacting protein, in a yeast two-hybrid library screen. OBF4 interacts with the B-box region of CO. The abundance of OBF4 mRNA cycles with a 24 h rhythm under both long-day (LD) and short-day (SD) conditions, with significantly higher levels during the night than during the day. Electrophoretic mobility shift assays revealed that OBF4 binds to the promoter of the FLOWERING LOCUS T (FT) gene, a direct target of CO. We also found that, like CO and FT, an OBF4:GUS construct was prominently expressed in the vascular tissues of leaf, indicating that OBF4 can regulate FT expression through the formation of a protein complex with CO. Taken together, our results suggest that OBF4 may act as a link between defense responses and flowering.

• Molecules and Cells
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• v.42 no.12
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• pp.858-868
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• 2019

Shoot branching is an essential agronomic trait that impacts on plant architecture and yield. Shoot branching is determined by two independent steps: axillary meristem formation and axillary bud outgrowth. Although several genes and regulatory mechanism have been studied with respect to shoot branching, the roles of chromatin-remodeling factors in the developmental process have not been reported in rice. We previously identified a chromatin-remodeling factor OsVIL2 that controls the trimethylation of histone H3 lysine 27 (H3K27me3) at target genes. In this study, we report that loss-of-function mutants in OsVIL2 showed a phenotype of reduced tiller number in rice. The reduction was due to a defect in axillary bud (tiller) outgrowth rather than axillary meristem initiation. Analysis of the expression patterns of the tiller-related genes revealed that expression of OsTB1, which is a negative regulator of bud outgrowth, was increased in osvil2 mutants. Chromatin immunoprecipitation assays showed that OsVIL2 binds to the promoter region of OsTB1 chromatin in wild-type rice, but the binding was not observed in osvil2 mutants. Tiller number of double mutant osvil2 ostb1 was similar to that of ostb1, suggesting that osvil2 is epistatic to ostb1. These observations indicate that OsVIL2 suppresses OsTB1 expression by chromatin modification, thereby inducing bud outgrowth.

• Journal of Plant Biotechnology
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• v.40 no.3
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• pp.169-177
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• 2013

Recently, the number of people with diabetes is rapidly increasing, coupled with the fact that the insulin market is remarkably increasing. Therefore, molecular farming for plant-derived pharmaceutical protein production is reported as becoming more attractive than ever. In this study, we carried out experiments step by step for development of recombinant insulin constructs, which were transformed into E. coli system, in vitro transcription and translation system, and tobacco cells. At first, recombinant proinsulin protein was successfully produced in in vitro transcription and translation system with wheat germ extract. After which, recombinant construct of prominiinsulin encoded a fusion protein of 7.8 kDa with trypsin cleavage sites at N terminus and C terminus of minimized C-peptide was tried to in vitro expression using E.coli culture. After purification with His-tag column, the resulting recombinant prominiinsulin protein was processed with trypsin, and then checked insulin biosynthesis by SDS-PAGE and western blot analysis with anti-insulin monoclonal antibody. The immunoreactive product of trypsin-treated miniinsulin was identical to the predicted insulin hexamer. The construct of 35S promoter-driven preprominiinsulin recombinant gene with signal peptide region for ER-targeting and red fluorescence protein gene [N terminus ${\rightarrow}$ tobacco E2 signal peptide ${\rightarrow}$ B-peptide (1-29 AA) ${\rightarrow}$ AAK ${\rightarrow}$ A-peptide (1-21 AA) ${\rightarrow}$ RR ${\rightarrow}$ His6 ${\rightarrow}$ KDEL ${\rightarrow}$ C terminus] was transformed into BY-2 tobacco cells. A polypeptide corresponding to the 38-kDa molecular mass predicted for fusion protein was detected in total protein profiles from transgenic BY-2 cells by western analysis. Therefore, this recombinant preprominiinsulin construct can be used for generation of transgenic tobacco plants producing therapeutic recombinant insulin.

• Food Science and Biotechnology
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• v.18 no.5
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• pp.1204-1211
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• 2009

Hericium erinaceus is an edible mushroom used as a medicinal food in Asian countries. In this study, the chemopreventive effects of H. erinaceus mycelia hot water extract (HEW) were evaluated. HEW remarkably induced the luciferase activity of the antioxidant response element (ARE), located in the promoter region of phase 2 and antioxidant genes and regulated by nuclear factor E2-related factor 2 (Nrf2). The up-regulation of ARE activity by HEW corresponded with the induction of Nrf2 and the antioxidant enzyme, hemeoxygenase-1. The inhibition of cyclooxygenase-2 (COX-2) activity is a promising effective approach in cancer chemoprevention, and HEW prominently suppressed COX-2 protein expression in HepG2 cells. Furthermore, HEW showed anti-inflammatory activity by modulating inflammatory mediators such as nitric oxide (NO), inducible NO synthase, tumor necrosis factor-${\alpha}$, interleukin-$1{\beta}$, and the transcription factor, nuclear factor-${\kappa}B$, in lipopolysaccharide-stimulated RAW 264.7 cells. These results suggest that H. erinaceus possessed anti-tumor and anti-inflammatory effects via the modulation of Nrf2/ARE and inflammatory signaling pathways, and may therefore have potential use as a natural chemopreventive agent.

• Clinical and Experimental Reproductive Medicine
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• v.23 no.3
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• pp.269-275
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• 1996

Biosynthesis and secretion of anterior pituitary hormones are under the control of specific hypothalamic stimulatory and inhibitory factors. Among them, Growth Hormone Releasing Hormone (GHRH) is the major stimulator of pituitary somatotrophs activating GH gene expression and secretion. Human GHRH is a polypeptide of 44 amino acids initially isolated from pancreatic tumors, and the gene for the hypothalamic form of GHRH is organized into 5 exons spanning over 10 kilobases (kb) on genomic DNA and encodes a messenger RNA of 700-750 nucleotides. Several neuropeptides classically associated with the hypothalamus have been found in the extrahypothalamic regions, suggesting the existence of novel sources, targets and functions. GHRH-like immunoreactivity has been found in several peripheral sites, including placenta, testis, and ovary, indicating that GHRH may also have regulatory roles in peripheral reproductive organs. Furthermore, higher molecular weight forms of the GHRH transcripts were identified from these organs (1.75 kb in testis; 1.75 and >3 kb in ovary). These tissue-specific expression of GHRH gene suggest the existence of unique regulatory mechanism of GHRH expression and function in these organs. In fact, placenta-specific and testis-specific promoters for GHRH transcripts which are located in about 10 kb upstream region of hypothalamic promoter were reported. The use of unique promoters in extrahypothalamic sites could be refered in a different control of GHRH gene and different functions of the translated products in these tissues. Somatotrophs and lactotrophs have been thought to be derived from a common bipotential progenitor, the somatolactotrophs, which give origins to either phenotypes. Although the precise mechanism responsible for the lactotroph differentiation in the anterior pituitary gland has not been yet clalified, there are several candidators for the generation of lactotrophs. In human, the presence of GHRH peptides with different size from authentic hypothalamic form in the normal anterior pituitary and several types of adenoma were demonstrated. Recently our group found the existence of immunoreactive GHRH and its transcript from the normal rat anterior pituitary (gonadotroph> somatotroph> lactotroph), and the GHRH treatment evoked the increased proliferation rate of anterior pituitary cells in vitro. The transgenic mouse models clearly shown that GHRH or NGF overexpression by anterior pituitary cells induced development of pituitary hyperplasia and adenomas particularly GH-oma and prolactinoma. Taken together, we hypothesize that the pituitary GHRH could serve not only as a modulator of hormone secretion but as a paracrine or autocrine regulator of anterior pituitary cell proliferation and differentiation. Interestingly enough, the expression of Pit-1 homeobox gene (the POU class transcription factor) was confined to somatotrophs, lactotrophs and somatolactotrophs in which GHRH receptors are expressed commonly. Concerning the mechanism of somatolactotroph and lactotroph differentiation in the anterior pituitary, we have focused following two possibilities; (1) changes in the relative levels or interactions of both hypothalamic and intrapituitary factors such as dopamine, VIP, somatostatin, NGF and GHRH; (2) alterations of GHRH-GHRH receptor signaling and Pit-1 activity may be the cause of lactotroph differentiation or pituitary hyperplasia and adenoma formation. Extensive further studies will be necessary to solve these complicated questions.

• Korean Journal of Microbiology
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• v.36 no.4
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• pp.254-258
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• 2000

Invasion process of bacterial cell into intestinal epithelium is important in Salmonella infection. The invasion is induced by the proteins secreted by type III secretion appratus of Salmonella. It has been known that the proteins do not have N-terminal signal peptide existing in general secreted proteins. Recent studies on Yersinia reported that secretion signal of type III appratus may lie on 5'end secondary structure of mRNA of secreted protein. In this study, we constructed translational fusion of ompR and sopE, encoding type III secretion protein of Salmonella, and observed secretion of the fusion protein for investigating the secretion signal of Salmonella type III appratus. The sopE DNA fragments of the translational fusion contain the region of promoter and from start code to tenth or to fifth code. These translational fusions indicate that type III secretion signal of Salmonella is located on 5'end of mRNA encoding secreted protein. We constructed prototype of secretion vector using this signal to produce useful foreign protein.

• Journal of Microbiology
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• v.44 no.3
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• pp.301-310
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• 2006

Two pathways of ammonium assimilation and glutamate biosynthesis have been identified in microorganisms. One pathway involves the NADP-linked glutamate dehydrogenase, which catalyzes the amination of 2-oxoglutarate to form glutamate. An alternative pathway involves the combined activities of glutamine synthetase, which aminates glutamate to form glutamine, and glutamate synthase, which transfers the amide group of glutamine to 2-oxoglutarate to yield two molecules of glutamate. We have cloned the large subunit of the glutamate synthase (GOGAT) from Salmonella typhimurium by screening the expression of GOGAT and complementing the gene in E. coli GOGAT large subunit-deficient mutants. Three positive clones (named pUC19C12, pUC19C13 and pUC19C15) contained identical Sau3AI fragments, as determined by restriction mapping and Southern hybridization, and expressed GOGAT efficiently and constitutively using its own promoter in the heterologous host. The coding region expressed in Escherichia coli was about 170 kDa on SDS-PAGE. This gene spans 4,732 bases, contains an open reading frame of 4,458 nucleotides, and encodes a mature protein of 1,486 amino acid residues (Mr =166,208). The EMN-binding domain of GOGAT contains 12 glycine residues, and the 3Fe-4S cluster has 3 cysteine residues. The comparison of the translated amino acid sequence of the Salmonella GOGAT with sequences from other bacteria such as Escherichia coli, Salmonella enterica, Shigella flexneri, Yersinia pestis, Vibrio vulnificus and Pseudomonas aeruginosa shows sequence identity between 87 and 95%.