• Clinical and Experimental Reproductive Medicine
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• v.42 no.2
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• pp.58-61
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• 2015

Objective: The XIST gene is considered to be an attractive candidate gene for skewed X-chromosome inactivation and a possible cause of primary ovarian insufficiency (POI). The purpose of this study was to investigate whether the XIST gene promoter mutation is associated with idiopathic POI in a sample of the Korean population. Methods: Subjects consisted of 102 idiopathic POI patients and 113 healthy controls with normal menstrual cycles. Patients with the following known causes of POI were excluded in advance: cytogenetic abnormalities, prior chemo- or radiotherapy, or prior bilateral oophorectomy. Genotyping was performed using polymerase chain reaction-restriction fragment length polymorphism analysis. Results: The mean age of onset of ovarian insufficiency was $28.7{\pm}8.5years$ and the mean values of serum luteinizing and follicle-stimulating hormones and estradiol in the POI group were $31.4{\pm}18.2mIU/mL$, $74.5{\pm}41.1mIU/mL$, and $30.5{\pm}36.7pg/mL$, respectively. We found no cytosine to guanine (C43G) variation in the XIST gene in both POI patients and controls. Conclusion: The C43G mutation in the promoter region of the XIST gene was not present in the Korean patients with idiopathic POI in our study, in contrast to our expectation, suggesting that the role of XIST in the pathogenesis of POI is not yet clear.

• BMB Reports
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• v.35 no.6
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• pp.553-561
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• 2002

A gene that encodes a protein homologue to baculoviral IE-1 was identified and sequenced in the genome of the Choristoneura fumiferana granulovirus (ChfuGV). The gene has an 1278 nucleotide (nt) open-reading frame (ORF) that encodes 426 amino acids with an estimated molecular weight of 50.33 kDa. At the nucleotide level, several cis-acting regulatory elements were detected within the promoter region of the ie-1 gene of ChfuGV along with other studied granuloviruses (GVs). Two putative CCAAT elements were detected within the noncoding leader region of this gene; one was located on the opposite strand at -92 and the other at -420 nt from the putative start triplet. Two baculoviral late promoter motifs (TAAG) were also detected within the promoter region of the ie-1 gene of ChfuGV. A single polyadenylation signal, AATAAA, was located 18nt downstream of the putative translational stop codon of ie-1 from ChfuGV. At the protein level, the amino acid sequence data that was derived from the nucleotide sequence in ChfuGV IE-1 was compared to those of the Cydia pomonella granulovirus (CpGV), Xestia c-nigrum granulovirus (XcGV) and Plutella xylostella granulovirus (PxGV). The C-terminal regions of the granuloviral IE-1 sequences appeared to be more conserved when compared to the N-terminal regions. A domain, similar to the basic helix-loop-helix like (bHLH-like) domain in NPVs, was detected at the C-terminal region of IE-1 from ChfuGV (residues 387 to 414). A phylogenetic tree for baculoviral IE-1 was constructed using a maximum parsimony analysis. A phylogenetic estimation demonstrates that ChfuGV IE-1 is most closely related to that of CpGV.

• BMB Reports
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• v.46 no.9
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• pp.454-459
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• 2013

LRRK2 (leucine-rich repeat kinase 2) has been identified as a gene corresponding to PARK8, an autosomal-dominant gene for familial Parkinson's disease (PD). LRRK2 pathogenic-specific mutants induce neurotoxicity and shorten neurites. To elucidate the mechanism underlying LRRK2 expression, we constructed the LRRK2-promoter-luciferase reporter and used it for promoter analysis. We found that the glucocorticoid receptor (GR) transactivated LRRK2 in a ligand-dependent manner. Using quantitative RT-PCR and Western analysis, we further showed that treatment with dexamethasone, a synthetic GR ligand, induced LRRK2 expression at both the transcriptional and translational levels, in dopaminergic MN9D cells. Dexamethasone treatment also increased expression of ${\alpha}$-synuclein, another PD causative gene, and enhanced transactivation of the ${\alpha}$-synuclein promoter-luciferase reporter. In addition, dexamethasone treatment to MN9D cells weakly induced cytotoxicity based on an LDH assay. Because glucocorticoid hormones are secreted in response to stress, our data suggest that stress might be a related factor in the pathogenesis of PD.

• Asian Pacific Journal of Cancer Prevention
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• v.17 no.1
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• pp.219-223
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• 2016

Background: Promoter hypermethylation is a frequent epigenetic mechanism for gene transcription repression in cancer and is one of the hallmarks of the disease. Cadherin EGF LAG seven-pass G-type receptor 3 (CELSR3) contributes to cell contact-mediated communication. Dysregulation of promoter methylation has been reported in various cancers. Objectives: The objectives of this study were to investigate the CELSR3 hypermethylation level in oral squamous cell carcinomas (OSCCs) using methylation-sensitive high-resolution melting analysis (MS-HRM) and to correlate CELSR3 methylation with patient demographic and clinicopathological parameters. Materials and Methods: Frozen tissue samples of healthy subjects' normal mucosa and OSCCs were examined with regard to their methylation levels of the CELSR3 gene using MS-HRM. Results: MS-HRM analysis revealed a high methylation level of CELSR3 in 86% of OSCC cases. Significant correlations were found between CELSR3 quantitative methylation levels with patient ethnicity (P=0.005), age (P=0.024) and pathological stages (P=0.004). A moderate positive correlation between CELSR3 and patient age was also evident (R=0.444, P=0.001). Conclusions: CELSR3 promoter hypermethylation may be an important mechanism involved in oral carcinogenesis. It may thus be used as a biomarker in OSCC prognostication.

• BMB Reports
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• v.39 no.6
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• pp.730-736
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• 2006

To evaluated the effect of recombinant adenovirus Ad-ODC-AdoMetDCas which can simultaneously express both antisense ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (AdoMetDC) on cell cycle distribution in colorectal cancer cell and investigated underlying regulatory responses, human colorectal cancer cells HT-29 were cultured in RPMI 1640 medium and infected with Ad-ODC-AdoMetDCas. Cell cycle progression was detected by flow cytometry analysis. The expression levels of cell cycle regulated proteins were measured by Western blot analysis. The mRNA level of cyclin D1 was measured by RT-PCR. And a luciferase reporter plasmid of cyclin D1 promoter was constructed to observe the effect of Ad-ODC-AdoMetDCas on cyclin D1 promoter activity. The results showed that recombinant adenovirus Ad-ODC-AdoMetDCas significantly induced $G_1$ arrest, decreased levels of cyclin D1 protein and mRNA and suppressed the promoter activity. Ad-ODC-AdoMetDCas also inhibited nuclear translocation of $\beta$-catenin. In conclusion, downregulation of ODC and AdoMetDC mediated by Ad-ODC-AdoMetDCas transfection induces $G_1$ arrest in HT-29 cells and the arrest was associated with suppression of cyclin D1 expression and inhibition of $\beta$-catenin nuclear translocation. As a new anticancer reagent, the recombinant adenovirus Ad-ODC-AdoMetDCas holds promising hope for the therapy of colorectal cancers.

• Fisheries and Aquatic Sciences
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• v.18 no.2
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• pp.183-193
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• 2015

Several transgenic marine medaka Oryzias dancena strains harboring a green fluorescent protein (GFP) reporter construct regulated by an endogenous ${\beta}$-actin promoter were established and their expression characteristics in relation to transgene copy numbers were examined in 21 transgene genotypes. Most of the transgenic strains displayed transgene insertion patterns typical of microinjection-mediated introduction of foreign DNA into fish embryos, characterized by the random integration of multiple transgene copies (ranging from 1 - 282 copies per cell), often accompanied by the formation of concatemer(s), as assessed by genomic Southern blot hybridization analysis and qPCR. Transgenic strains showed ubiquitous and continued temporal and spatial expression patterns of the transgenic GFP during most of their life cycle, from the embryonic stage to adulthood, enabling assessment of the expression pattern of the endogenous ${\beta}$-actin gene. However, a comparative evaluation of transgene copy numbers and expression levels showed that copy number-dependent expression, the stability of the ubiquitous distribution and expression efficiency per transgene copy varied among the transgenic strains. Fluorescence expression levels were positively correlated with absolute transgene copy numbers, whereas the expression efficiency per transgene copy was inversely related to the number of transgene integrant copies. Data from this study will guide the selection of potentially desirable transgenic strains with ubiquitous expression of a fluorescent transgene, not only in this marine medaka species but also in other related model fish species.

• Journal of Plant Biotechnology
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• v.31 no.2
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• pp.175-178
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• 2004

Transgenic tobacco (Nicotiana tabacum L. cv Bright Yellow-2) cell lines expressing a human histone H1.5 (referred to as hH1.5), which suppress collagen-induced rheumatoid arthritis, were developed under the oxidative stress-inducible peroxidase (SWPA2) promoter. Tobacco BY-2 cells were transformed by Agrobacterium-mediated method. The kanamycin-resistant calli were selected on the modified MS medium containing 150mg/L kanamycin and 300mg/L claforan. Transgenic cell lines were confirmed by PCR and northern blot analysis. Recombinant hH1.5 (rhH1.5) protein (42 kDa) was also detected by Western blot analysis, showing a different molecular weight of human hH1.5 (32 kDa). These results suggested that a hH1.5 gene was properly introduced in tobacco cultured cells under the control of SWPA2 promoter. The further characterization of rhH1.5 protein remains to be studied.

• Journal of Plant Biology
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• v.37 no.1
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• pp.77-83
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• 1994

DNA uptake in dry embryos of rice by DNA imbibition was detected by monitoring the expression of chimeric vectors. The selective markers of expression vectors used were ${\beta}-glucuronidase$ ronidase (GUS) and hygromycin phosphotransferase (HPT) genes under the control of CaMV35 S promoter. Frequency of transient expression of the foreign gene was generally 30-50% varying according to the types of vectors and rice cultivars. Dot blot analysis and DNA sequence analysis of inverse polymerase chain reaction products showed that selected rice in hygromycin B (HmB) medium had HPT gene and CaMV35S promoter DNA sequence in genomic DNA of rice. To investigate what ratio of rice having two marker genes simultaneously as rice embryos imbibed the vector DNA having two HPT and GUS gene, transform ants selected in lImB medium were subjected to PCR for GUS gene. It was shown that about 90 percentage of surviving ones in HmB medium had GUS gene.S gene.

• Molecules and Cells
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• v.40 no.8
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• pp.577-586
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• 2017

Phytocystatins (PhyCYSs) are plant-specific proteinaceous inhibitors that are implicated in protein turnover and stress responses. Here, we characterized a PhyCYS from Arabidopsis thaliana, which was designated AtCYS5. RT-qPCR analysis showed that the expression of AtCYS5 in germinating seeds was induced by heat stress (HS) and exogenous abscisic acid (ABA) treatment. Analysis of the expression of the ${\beta}-glucuronidase$ reporter gene under the control of the AtCYS5 promoter showed that AtCYS5 expression during seed germination was induced by HS and ABA. Constitutive overexpression of AtCYS5 driven by the cauliflower mosaic virus 35S promoter led to enhanced HS tolerance in transgenic Arabidopsis, which was characterized by higher fresh weight and root length compared to wild-type (WT) and knockout (cys5) plants grown under HS conditions. The HS tolerance of AtCYS5-overexpressing transgenic plants was associated with increased insensitivity to exogenous ABA during both seed germination and post-germination compared to WT and cys5. Although no HS elements were identified in the 5'-flanking region of AtCYS5, canonical ABA-responsive elements (ABREs) were detected. AtCYS5 was upregulated in ABAtreated protoplasts transiently co-expressing this gene and genes encoding bZIP ABRE-binding factors (ABFs and AREB3). In the absence of ABA, ABF1 and ABF3 directly bound to the ABREs in the AtCYS5 promoter, which activated the transcription of this gene in the presence of ABA. These results suggest that an ABA-dependent pathway plays a positive role in the HS-responsive expression of AtCYS5 during seed germination and post-germination growth.

• Asian-Australasian Journal of Animal Sciences
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• v.32 no.6
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• pp.891-895
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• 2019

Objective: Aim of present study was the set up of a fast and reliable protocol using species-specific markers for the quali-quantitative analysis of DNA and the detection of ruminant biological components in dairy products. For this purpose, the promoter of the gene coding for the ${\alpha}$-lactoalbumin (LALBA) was chosen as possible candidate for the presence of short interspersed nuclear elements (SINEs). Methods: DNA was isolated from somatic cells of 120 individual milk samples of cattle (30), Mediterranean river buffalo (30), goat (30), and sheep (30) and the gene promoter region (about 600/700 bp) of LALBA (from about 600 bp upstream of exon 1) has been sequenced. For the development of a single polymerase chain reaction (PCR) protocol that allows the simultaneous identification of DNA from the four species of ruminants, the following internal primers pair were used: 5'-CACTGATCTTAAAGCTCAGGTT-3' (forward) and 5'-TCAGA GTAGGCCACAGAAG-3' (reverse). Results: Sequencing results of LALBA gene promoter region confirmed the presence of SINEs as monomorphic "within" and variable in size "among" the selected species. Amplicon lengths were 582 bp in cattle, 592 bp in buffalo, 655 in goat and 729 bp in sheep. PCR specificity was demonstrated by the detection of trace amounts of species-specific DNA from mixed sources ($0.25ng/{\mu}L$). Conclusion: We developed a rapid PCR protocol for the quali-quantitative analysis of DNA and the traceability of dairy products using a species-specific marker with only one pair of primers. Our results validate the proposed technique as a suitable tool for a simple and inexpensive (economic) detection of animal origin components in foodstuffs.