• 식물병연구
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• 제30권2호
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• pp.157-164
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• 2024

Jack bean (Canavalia ensiformis) is one of healthy products for fermented or functional food in Korea and is widely distributed and cultivated worldwide. During August 2022, Jack bean plants showing symptoms of yellow flecks, chlorosis, necrotic spots and mosaic were observed in Jangheung-gun, South Korea. By transmission electron microscopy, flexuous filamentous virus particles of approximately 750×13 nm in size were observed in the symptomatic leaf samples. The infection of a Korean isolate of clover yellow vein virus (ClYVV-Ce-JH) was confirmed using double antibody sandwich enzyme-linked sorbent assay, reverse transcription polymerase chain reaction and high-throughput sequencing. The complete genome sequence of ClYVV-Ce-JH consists of 9,549 nucleotides (nt) excluding the poly (A) tail and encodes 3,072 amino acids (aa), with an AUG start and UAG stop codon, containing one open reading frame that is typical of a potyvirus polyprotein. The polyprotein of ClYVV-Ce-JH was divided into ten proteins and each protein's cleavage sites were determined. The coat protein (CP) and polyprotein of ClYVV-Ce-JH were compared at the nt and aa levels with those of the previously reported 14 ClYVV isolates. ClYVV-Ce-JH shared 92.62% to 99.63% and 93.39% to 98.05% at the CP and polyprotein homology. To our knowledge, this is the first report of a Korean isolate of ClYVV from Jack bean plants and the complete genome sequence of a ClYVV Jack bean isolate in the world.

• 한국발생생물학회:학술대회논문집
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• 한국발생생물학회 2003년도 제3회 국제심포지움 및 학술대회
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• pp.81-81
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• 2003

DNA methylation is a covalent modification of DNA that can modulate gene expression and is now recognized as a major component of the epigenome. During evolution, the dinucleotide CpG has been progressively eliminated from the genome of higher eukaryotes and is present at only 5% to 10% of its predicted frequency. Approxymately 80% of the remaining CpG sites contain methylated cytosines in most vertebrates and they are distributed in a pattern that is unique in each tissue and is inversely correlated with gene expression. The pattern of methylation is faithfully maintained during cell division by the enzyme Dnmt1, the maintenance DNA methyltransferase, which catalyzes the transfer of a methyl group from S-adenosyl-methionine to the 5'-position of the cytosine ring. We have been identified bovine Dnmt1 cDNA full-length recently (AY173048) Little is known on the functions of Dnmt1 in bovine preimplantation embryos. Thus, we analyzed the specific pattern of Dnmt1 in in vitro derived/nuclear transfer bovine and in vivo derived mouse embryos to monitor the epigenetic reprogramming process. We investigated these process by using indirect immunofluresence with an antibody to Dnmt1. According to other studies, Dnmt1 accumulates in nuclei of early growing oocytes but is sequestered in the cytoplasm of mature oocytes. In 2-cell and 4-cell embryos, Dnmt1 is cytoplasmic, but at the 8-cell stage, it is present only in the nucleus. By the blastocyst stage, Dnmt1o is again found only in the cytoplasm. Thus, nuclear localization of Dnmt1o in preimplantation embryos is limited to the 8-cell stages After implantation, Dnmt1 is localized in the nucleus in mouse. However, we have found different patterns of Dnmt1 nuclear localization. Though we used the common antibody, immune-localization data revealed that Dnmt1 antibody have been detected at the nucleus in 1-cell to blastocyst embryos. Therefore, maybe we think that the functions of Dnmt1 between bovine and mice are different. In order to Identify the mechanisms that regulate DNA methylation in bovine preimplantation embryo, we have plans on using bovine oocyte and somatic specific Dnmt1 antibodies.

• Asian-Australasian Journal of Animal Sciences
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• 제21권11호
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• pp.1572-1575
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• 2008

The xenotransplantation of pig organs and cells can be related with a risk of transmission of infectious diseases to human. Previous findings indicate that the regulatory region of PERV for retroviral transcription, replication and integration into the cellular DNA is located on the 5' Long Terminal Repeat (LTR). The objective of this study is the investigation of methylation and deletion status of the PERV 5' LTR region which can be used for regulating PERV expression. We compared the sequences of genomic DNA and bisulfite-treated genomic DNA from PK-15 cells expressing PERV to observe the methylation status of the 5' LTR. Our results showed that the CpG sites of U3 were methylated and methylation was inconsistent in the R and U5 regions. Also, variable numbers of 18 bp repeats and 21 bp repeats were detected on 5' LTR by sequencing analysis. The consistent U3 methylation might be indicative of host suppression of expression of the retroviruses.

• 응용통계연구
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• 제29권6호
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• pp.1117-1128
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• 2016

고차원 유전체 자료를 사용하는 유전체 연관 분석에서는 벌점 우도함수 기반의 회귀계수 규제화 방법이 질병 및 표현형질에 영향을 주는 유전자를 발견하는데 많이 이용된다. 특히, 네트워크 기반의 규제화 방법은 유전체 연관성 연구에서의 유전체 경로나 신호 전달 경로와 같은 생물학적 네트워크 정보를 사용할 수 있으므로, Lasso나 Elastic-net과 같은 다른 규제화 방법들과 비교했을 경우 네트워크 기반의 규제화 방법이 보다 더 정확하게 관련 유전자들을 찾아낼 수 있다는 장점을 가지고 있다. 그러나 네트워크 기반의 규제화 방법은 그룹 구조를 갖고 있는 고차원 유전체 자료에는 적용시킬 수 없다는 문제점을 가지고 있다. 실제 SNP 데이터와 DNA 메틸화 데이터처럼 대다수의 고차원 유전체 자료는 그룹 구조를 가지고 있으므로 본 논문에서는 이러한 그룹 구조를 가지고 있는 고차원 유전체 자료를 분석하고자 네트워크 기반의 규제화 방법에 주성분 분석(principal component analysis; PCA)과 부분 최소 자승법(partial least square; PLS)과 같은 차원 축소 방법을 결합시키는 새로운 분석 방법을 제안하고자 한다. 새롭게 제안한 분석 방법은 몇 가지의 모의실험을 통해 변수 선택의 우수성을 입증하였으며, 또한 152명의 정상인들과 123명의 난소암 환자들로 구성된 고차원 DNA 메틸화 자료 분석에도 사용하였다. DNA 메틸화 자료는 대략 20,000여개의 CpG sites가 12,770개의 유전자에 포함되어 있는 그룹 구조를 가지고 있으며 Illumina Innium uman Methylation27 BeadChip으로부터 생성되었다. 분석 결과 우리는 실제로 암에 연관된 몇 가지의 유전자를 발견할 수 있었다.

• Maxillofacial Plastic and Reconstructive Surgery
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• 제22권2호
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• pp.164-173
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• 2000

The p16 protein is a cyclin dependent kinase inhibitor that inhibits cell cycle progression from $G_1$ phase to S phase in cell cycle. Many p16 gene mutations have been noted in many cancer-cell lines and in some primary cancers, and alterations of p16 gene function by DNA methylation have been noticed in various kinds of cancer tissues and cell-lines. There have been a large body of literature has accumulated indicating that abnormal patterns of DNA methylation (both hypomethylation and hypermethylation) occur in a wide variety of human neoplasma and that these aberrations of DNA methylation may play an important epigenetic role in the development and progression of neoplasia. DNA methylation is a part of the inheritable epigenetic system that influences expression or silencing of genes necessary for normal differentiation and proliferation. Gene activity may be silenced by methylation of up steream regulatory regions. Reactivation is associated with demethylation. Although evidence or a high incidence of p16 alterations in a variety of cell lines and primary tumors has been reported, that has been contested by other investigators. The precise mechanisms by which abnormal methylation might contribute to carcinogenesis are still not fully elucidated, but conceivably could involve the modulation of oncogene and other important regulatory gene expression, in addition to creating areas of genetic instability, thus predisposing to mutational events causing neoplasia. There have been many variable results of studies of head and neck squamous cell carcinoma(HNSCC). This investigation was studied on 13 primary HNSCC for p16 gene status by protein expression in immunohistochemistry, and DNA genetic/epigenetic analyzed to determine the incidence, the mechanisms, and the potential biological significance of its Inactivation. As methylation detection method of p16 gene, the methylation specific PCR(MSP) is sensitive and specific for methylation of any block of CpG sites in a CpG islands using bisulfite-modified DNA. The genomic DNA is modified by treatment with sodium bisulfate, which converts all unmethylated cytosines to uracil(thymidine). The primers designed for MSP were chosen for regions containing frequent cytosines (to distinguish unmodified from modified DNA), and CpG pairs near the 5' end of the primers (to provide maximal discrimination in the PCR between methylated and unmethylated DNA). The two strands of DNA are no longer complementary after bisulfite treatment, primers can be designed for either modified strand. In this study, 13 paraffin embedded block tissues were used, so the fragment of DNA to be amplified was intentionally small, to allow the assessment of methylation pattern in a limited region and to facilitate the application of this technique to samlples. In this 13 primary HNSCC tissues, there was no methylation of p16 promoter gene (detected by MSP and automatic sequencing). The p16 protein-specific immunohistochemical staining was performed on 13 paraffin embedded primary HNSCC tissue samples. Twelve cases among the 13 showed altered expression of p16 proteins (negative expression). In this study, The author suggested that low expression of p16 protein may play an important role in human HNSCC, and this study suggested that many kinds of genetic mechanisms including DNA methylation may play the role in carcinogenesis.

• BMB Reports
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• 제57권2호
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• pp.110-115
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• 2024

Alterations in DNA methylation play an important pathophysiological role in the development and progression of colorectal cancer. We comprehensively profiled DNA methylation alterations in 165 Korean patients with colorectal cancer (CRC), and conducted an in-depth investigation of cancer-specific methylation patterns. Our analysis of the tumor samples revealed a significant presence of hypomethylated probes, primarily within the gene body regions; few hypermethylated sites were observed, which were mostly enriched in promoter-like and CpG island regions. The CpG Island Methylator Phenotype-High (CIMP-H) exhibited notable enrichment of microsatellite instability-high (MSI-H). Additionally, our findings indicated a significant correlation between methylation of the MLH1 gene and MSI-H status. Furthermore, we found that the CIMP-H had a higher tendency to affect the right-side of the colon tissues and was slightly more prevalent among older patients. Through our methylome profile analysis, we successfully verified the methylation patterns and clinical characteristics of Korean patients with CRC. This valuable dataset lays a strong foundation for exploring novel molecular insights and potential therapeutic targets for the treatment of CRC.

• Journal of Microbiology and Biotechnology
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• 제20권6호
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• pp.1022-1026
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• 2010

The different cleavage patterns of pYBamy59 plasmid isolated from E. coli $DH5{\alpha}$ and B. longum MG1 by the cell extract of B. longum MG1 suggested that the main reason for its low transformation efficiency was related to the restriction modification (R-M) system. To confirm the correlation between the R-M system and transformation efficiency, in vitro methylation and site-directed mutagenesis were performed in pYBamy59. Sequence analysis of pYBamy59 fragments digested by the cell extract of B. longum MG1 revealed that all fragments were generated by restriction of the sequence recognized by SacII endonuclease. When pYBamy59 from E. coli was methylated in vitro by CpG or GpC methyltransferase, it was protected from SacII digestion. Site-directed mutagenesis, which removed SacII sites from pYBamy59, or in vitro methylation of pYBamy59 showed 8- to 15-fold increases in the transformation efficiency over intact pYBamy59. Modification of the SacII-related R-M system in B. longum MG1 and in vitro methylation in pYBamy 59 can improve the transformation efficiency in this strain. The results showed that the R-M system is a factor to limit introduction of exogenous DNA, and in vitro modification is a convenient method to overcome the barrier of the R-M system for transformation.

• 한국가축번식학회지
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• 제27권4호
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• pp.309-315
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• 2003

DNA methylation at CpG sites, which is a epigenetic modification, is associated with gene expression without change of DNA sequences. During early mouse embryogenesis, dynamic changes of DNA methylation occur. In this study, DNA methylation patterns of porcine embryos produced in vivo and in vitro were examined at various developmental stages by the immunocytochemical staining method. Interestingly, active demethylation was not observed on the paternal pronucleus of porcine zygotes. However, differences were detected in the passive demethylation process between in vivo and in vitro embryos. There was no change in the DNA methylation state until the blastocyst stage of in vivo embryos, whereas partial demethylation was observed in several blastomeres from a 4 cell stage to a morula stage of in vitro embryos. The whole genome of inner cell mass (ICM) and trophectoderm (TE) cells in porcine blastocysts were evenly methylated without de novo methylation. Our findings demonstrate that genome-wide demethylation does not occur in pig embryos during preimplantation development unlike murine and bovine embryos. It indicates that the machinery regulating epigenetic reprogramming may be different between species.

• Asian Pacific Journal of Cancer Prevention
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• 제15권22호
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• pp.9797-9800
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• 2014

Background: Nasopharyngeal carcinoma (NPC) is a common cancer in Southern China and Southeast Asia. Alu elements are among the most prevalent repetitive sequences and constitute 11% of the human genome. Although Alu methylation has been evaluated in many types of cancer, few studies have examined the levels of this modification in serum from NPC patients. Objective: To compare the Alu methylation levels and patterns between serum from NPC patients and normal controls. Materials and Methods: Sera from 50 NPC patients and 140 controls were examined. Quantitative combined bisulfite restriction analysis-Alu (qCOBRA-Alu) was applied to measure Alu methylation levels and characterize Alu methylation patterns. Amplified products were classified into four patterns according to the methylation status of 2 CpG sites: hypermethylated (methylation at both loci), partially methylated (methylation of either of the two loci), and hypomethylated (unmethylated at both loci). Results: A comparison of normal control sera with NPC sera revealed that the latter presented a significantly lower methylation level (p=0.0002) and a significantly higher percentage of hypomethylated loci (p=0.0002). The sensitivity of the higher percentage of Alu hypomethyted loci for distinguishing NPC patients from normal controls was 96%. Conclusions: Alu elements in the circulating DNA of NPC patients are hypomethylated. Moreover, Alu hypomethylated loci may represent a potential biomarker for NPC screening.

• Asian Pacific Journal of Cancer Prevention
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• 제14권9호
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• pp.5495-5501
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• 2013

Background: Alu elements are one of the most common repetitive sequences that now constitute more than 10% of the human genome and potential targets for epigenetic alterations. Correspondingly, methylation of these elements can result in a genome-wide event that may have an impact in cancer. However, studies investigating the genome-wide status of Alu methylation in cancer remain limited. Objectives: Oral squamous cell carcinoma (OSCC) presents with high incidence in South-East Asia and thus the aim of this study was to evaluate the Alu methylation status in OSCCs and explore with the possibility of using this information for diagnostic screening. We evaluated Alu methylation status in a) normal oral mucosa compared to OSCC; b) peripheral blood mononuclear cells (PBMCs) of normal controls comparing to oral cancer patients; c) among oral epithelium of normal controls, smokers and oral cancer patients. Materials and Methods: Alu methylation was detected by combined bisulfite restriction analysis (COBRA) at 2 CpG sites. The amplified products were classified into three patterns; hypermethylation ($^mC^mC$), partial methylation ($^uC^mC+^mC^uC$), and hypomethylation ($^uC^uC$). Results: The results demonstrate that the $%^mC^mC$ value is suitable for differentiating normal and cancer in oral tissues (p=0.0002), but is not significantly observe in PBMCs. In addition, a stepwise decrease in this value was observed in the oral epithelium from normal, light smoker, heavy smoker, low stage and high stage OSCC (p=0.0003). Furthermore, receiver operating characteristic (ROC) curve analyses demonstrated the potential of combined $%^mC$ or $%^mC^mC$ values as markers for oral cancer detection with sensitivity and specificity of 86.7% and 56.7%, respectively. Conclusions: Alu hypomethylation is likely to be associated with multistep oral carcinogenesis, and might be developed as a screening tool for oral cancer detection.