• BMB Reports
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• v.52 no.6
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• pp.397-402
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• 2019

Middle East respiratory syndrome coronavirus (MERS-CoV) uses the spike (S) glycoprotein to recognize and enter target cells. In this study, we selected two epitope peptide sequences within the receptor binding domain (RBD) of the MERS-CoV S protein. We used a complex consisting of the epitope peptide of the MERS-CoV S protein and CpG-DNA encapsulated in liposome complex to immunize mice, and produced the monoclonal antibodies 506-2G10G5 and 492-1G10E4E2. The western blotting data showed that both monoclonal antibodies detected the S protein and immunoprecipitated the native form of the S protein. Indirect immunofluorescence and confocal analysis suggested strong reactivity of the antibodies towards the S protein of MERS-CoV virus infected Vero cells. Furthermore, the 506-2G10G5 monoclonal antibody significantly reduced plaque formation in MERS-CoV infected Vero cells compared to normal mouse IgG and 492-1G10E4E2. Thus, we successfully produced a monoclonal antibody directed against the RBD domain of the S protein which could be used in the development of diagnostics and therapeutic applications in the future.

• Reproductive and Developmental Biology
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• v.36 no.1
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• pp.7-12
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• 2012

DNA methyltransferase 1 (Dnmt1) gene contains three different isoform transcripts, Dnmt1s, Dnmt1o, and Dnmt1p, are produced by alternative usage of multiple first exons. Dnmt1o is specific to oocytes and preimplantation embryos, whereas Dnmt1s is expressed in somatic cells. Here we determined that porcine Dnmt1o gene had differentially methylated regions (DMRs) in 5'-flanking region, while those were not found in the Dnmt1s promoter region. The methylation patterns of the porcine Dnmt1o/Dnmt1s DMRs were investigated using bisulfite sequencing and pyrosequencing analysis through all preimplantation stages from one cell to blastocyst stage in in vivo or somatic cell nuclear transfer (SCNT). The Dnmt1o DMRs contained 8 CpG sites, which located in -640 bp to -30 bp upstream region from transcription start site of the Dnmt1o gene. The methylation status of 5 CpGs within the Dnmt1o DMRs were distinctively different at each stage from one-cell to blastocyst stage in the $in$ $vivo$ or SCNT, respectively. 55.62% methylation degree of the Dnmt1o DMRs in the $in$ $vivo$ was increased up to 84.38% in the SCNT embryo, moreover, $de$ $novo$ methylation and demethylation occurred during development of porcine embryos from the one-cell stage to the blastocyst stage. However, the DNA methylation states at CpG sites in the Dnmt1s promoter regions were hypomethylated, and dramatically not changed through one-cell to blastocyst stage in the $in$ $vivo$ or SCNT embryos. In the present study, we demonstrated that the DMRs in the promoter region of the porcine Dnmt1o was well conserved, contributing to establishment and maintenance of genome-wide patterns of DNA methylation in early embryonic development.

• BMB Reports
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• v.43 no.6
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• pp.400-406
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• 2010

Cancer/testis (CT) antigens exhibit highly tissue-restricted expression and are considered promising targets for cancer vaccines. Here we identified a novel CT gene ZNF645 which restrictively expresses in normal human testes and lung cancer patients (68.3%). To investigate the promoter methylation status of ZNF645, we carried out bisulfite genomic sequencing and found that the CpG island in its promoter was heavily methylated in normal lung tissues without the expression of ZNF645, whereas there was high demethylation in normal human testes and lung carcinoma tissues with its expression. Also ZNF645 could be remarkably activated in A549 and HEK293T cells treated by DNA demethylation agent 5'-aza-2'-deoxycytidine. And the dual luciferase assay revealed that the promoter activity of the ZNF645 was inhibited by methylation of the CpG island region. Therefore, we proposed that ZNF645 is a CT gene and activated in human testis and lung cancers by demethylation of its promoter region.

• Animal Bioscience
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• v.35 no.6
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• pp.847-857
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• 2022

Objective: The effects of maternal undernutrition during midgestation on muscle fiber histology, myosin heavy chain (MyHC) expression, methylation modification of myogenic factors, and the mammalian target of rapamycin (mTOR) signaling pathway in the skeletal muscles of prenatal and postnatal goats were examined. Methods: Twenty-four pregnant goats were assigned to a control (100% of the nutrients requirement, n = 12) or a restricted group (60% of the nutrients requirement, n = 12) between 45 and 100 days of gestation. Descendants were harvested at day 100 of gestation and at day 90 after birth to collect the femoris muscle tissue. Results: Maternal undernutrition increased (p<0.05) the fiber area of the vastus muscle in the fetuses and enhanced (p<0.01) the proportions of MyHCI and MyHCIIA fibers in offspring, while the proportion of MyHCIIX fibers was decreased (p<0.01). DNA methylation at the +530 cytosine-guanine dinucleotide (CpG) site of the myogenic factor 5 (MYF5) promoter in restricted fetuses was increased (p<0.05), but the methylation of the MYF5 gene at the +274,280 CpG site and of the myogenic differentiation (MYOD) gene at the +252 CpG site in restricted kids was reduced (p<0.05). mTOR protein signals were down-regulated (p<0.05) in the restricted offspring. Conclusion: Maternal undernutrition altered the muscle fiber type in offspring, but its relationship with methylation in the promoter regions of myogenic genes needs to be elucidated.

• The Plant Pathology Journal
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• v.15 no.5
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• pp.270-279
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• 1999

cDNAs complementary to the 3'-terminal regions of two potyvirus genomes were cloned and sequenced. The clone G7 contains one open reading frame (ORF) of 1,338 nucleotides and a 3' untranslated region (3'-UTR) of 403 nucleotides at the 3'-end excluding the 3'end poly(A) tail. The putative viral coat protein (CP) shows 55%-92% amino acid sequence homology to those of Allium potyviruses. The genome size of the virus was analyzed to be about 9.0 kb by Northern blot analysis. Five cDNA clones were screened out using GPV2 oligonucleotide as a probe. One of these clones, DEA72, which has a longest cDNA insert, contains one ORF of 1,459 nucleotides and a 3'-UTR of 590 nucleotides at the 3'-end excluding the 3'-end poly(A) tail. The putative viral CP shows 57%-88% amino acid sequence homologies to those of Allium potyviruses. The genome size of the virus was analyzed to be about 9.6 kb by Northern blot analysis. The results of immunoblot and Northern blot analyses suggest that almost all of the tested garlic plants showing mosaic or streak symptoms are infected with DEA72-potyvirus in variable degrees but rarely infected with G7-potyvirus in variable degrees but rarely infected with DEA72-potyvirus in variable degrees but rarely infected with G7-potyvirus. Immunoelectron microscopy using anti-DEA72 CP antibody shows that this potyvirus is about 750 nm long and flexuous rod shaped.

• Asian-Australasian Journal of Animal Sciences
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• v.31 no.1
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• pp.3-12
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• 2018

Objective: DNA methylation plays a major role in regulating the expression of genes related to traits of economic interest (e.g., weight gain) in livestock animals. This study characterized and investigated the functional inferences of genome-wide DNA methylome in the loin (longissimus dorsi) muscle (LDM) of swine. Methods: A total of 8.99 Gb methylated DNA immunoprecipitation sequence data were obtained from LDM samples of eight Duroc pigs (four pairs of littermates). The reference pig genome was annotated with 78.5% of the raw reads. A total of 33,506 putative methylated regions (PMR) were identified from methylated regions that overlapped at least two samples. Results: Of these, only 3.1% were commonly observed in all eight samples. DNA methylation patterns between two littermates were as diverse as between unrelated individuals (p = 0.47), indicating that maternal genetic effects have little influence on the variation in DNA methylation of porcine LDM. The highest density of PMR was observed on chromosome 10. A major proportion (47.7%) of PMR was present in the repeat regions, followed by introns (21.5%). The highest conservation of PMR was found in CpG islands (12.1%). These results show an important role for DNA methylation in species- and tissue-specific regulation of gene expression. PMR were also significantly related to muscular cell development, cell-cell communication, cellular integrity and transport, and nutrient metabolism. Conclusion: This study indicated the biased distribution and functional role of DNA methylation in gene expression of porcine LDM. DNA methylation was related to cell development, cell-cell communication, cellular integrity and transport, and nutrient metabolism (e.g., insulin signaling pathways). Nutritional and environmental management may have a significant impact on the variation in DNA methylation of porcine LDM.

• BMB Reports
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• v.40 no.2
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• pp.135-141
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• 2007

Conjugation of the methyl group at the fifth carbon of cytosines within the palindromic dinucleotide 5'-CpG-3' sequence (DNA methylation) is the best studied epigenetic mechanism, which acts together with other epigenetic entities: histone modification, chromatin remodeling and microRNAs to shape the chromatin structure of DNA according to its functional state. The cancer genome is frequently characterized by hypermethylation of specific genes concurrently with an overall decrease in the level of 5-methyl cytosine, the pathological implication of which to the cancerous state has been well established. While the latest genome-wide technologies have been applied to classify and interpret the epigenetic layer of gene regulation in the physiological and disease states, the epigenetic testing has also been seriously explored in clinical practice for early detection, refining tumor staging and predicting disease recurrence. This critique reviews the latest research findings on the use of DNA methylation in cancer diagnosis, prognosis and staging/classification.

• Tuberculosis and Respiratory Diseases
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• v.83 no.1
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• pp.1-13
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• 2020

For the past three decades, more than a thousand of genetic studies have been performed to find out the genetic variants responsible for the risk of asthma. Until now, all of the discovered single nucleotide polymorphisms have explained genetic effects less than initially expected. Thus, clarification of environmental factors has been brought up to overcome the 'missing' heritability. The most exciting solution is epigenesis because it intervenes at the junction between the genome and the environment. Epigenesis is an alteration of genetic expression without changes of DNA sequence caused by environmental factors such as nutrients, allergens, cigarette smoke, air pollutants, use of drugs and infectious agents during pre- and post-natal periods and even in adulthood. Three major forms of epigenesis are composed of DNA methylation, histone modifications, and specific microRNA. Recently, several studies have been published on epigenesis in asthma and allergy as a powerful tool for research of genetic heritability in asthma albeit epigenetic changes are at the starting point to obtain the data on specific phenotypes of asthma. In this presentation, we mainly review the potential role of DNA CpG methylation in the risk of asthma and its sub-phenotypes including nonsteroidal anti-inflammatory exacerbated respiratory diseases.

• Journal of Plant Biotechnology
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• v.47 no.1
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• pp.15-25
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• 2020

Allium L. is one of the largest genera of the Amaryllidaceae family, with more than 920 species including many economically important species used as vegetables, spices, medicines, or ornamental plants. Currently, DNA barcoding tools are being successfully used for the molecular taxonomy of Allium. A total of 46 Allium species were collected from their native areas, and DNA was extracted using the IBRC DNA extraction kit. We used specific primers to PCR amplify matK. DNA sequences were edited and aligned for homology, and a phylogenetic tree was constructed using the neighbor-joining method. The results show thymine (38.5%) was the most frequent and guanine (13.9%) the least frequent nucleotide. The matK regions of the populations were quite highly conserved, and the amount of C and CT was calculated at 0.162 and 0.26, respectively. Analysis of the nucleotide substitution showed C-T (26.22%) and A-G (8.08%) to have the highest and lowest percent, respectively. The natural selection process dN/dS was 1.16, and the naturality test results were -1.5 for Tajima's D and -1.19 for Fu's Fs. The NJ dendrogram generated three distinct clades: the first contained Allium austroiranicum and A. ampeloprasum; the second contained A. iranshahrii, A. bisotunense, and A. cf assadi; and the third contained A. rubellum and other species. In this study, we tested the utility of the matK region as a DNA barcode for discriminating Allium. species.

• Genomics & Informatics
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• v.9 no.4
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• pp.194-196
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• 2011

Promoter prediction is a very important problem and is closely related to the main problems of bioinformatics such as the construction of gene regulatory networks and gene function annotation. In this context, we developed an integrated promoter prediction program using hybrid methods, PromoterWizard, which can be employed to detect the core promoter region and the transcription start site (TSS) in vertebrate genomic DNA sequences, an issue of obvious importance for genome annotation efforts. PromoterWizard consists of three main modules and two auxiliary modules. The three main modules include CDRM (Composite Dependency Reflecting Model) module, SVM (Support Vector Machine) module, and ICM (Interpolated Context Model) module. The two auxiliary modules are CpG Island Detector and GCPlot that may contribute to improving the predictive accuracy of the three main modules and facilitating human curator to decide on the final annotation.