• Reproductive and Developmental Biology
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• 제33권2호
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• pp.93-98
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• 2009

In this study, bovine Dnmt1 cDNA was sequenced and detected Dnmt1 mRNA level in bovine tissues by northern blot, methylation pattern of genome by southern blot, specific localization of Dnmt1 in mouse and bovine preimplantation embryos by immunocytostaining and Dnmt1 protein level in ovary and testis by western blot. Bovine Dnmt1 cDNA sequence showed more homology with that of human than mouse and rat. The RNA level of Dnmt1 was 10 times higher expression in placenta than other tissues. This indicates that placenta was hypermethylated compared to others organs. The genomic DNA could not be cut by a specific restriction enzyme (HpaII) in placenta, lung and liver of bovine. It suggests that Dnmt1 in some somatic cells was already methylated. Dnmt1, which has the antibody epitope 1316~1616, was distributed in nucleus and cytoplasm including the stage of pronuclear stage and maturation of oocyte and gradually weaken to blastocyst stage compare to negative. In addition, Dnmt1 was strongly expressed in tetraploid embryo and cloned 8-cell than IVF 8-cell. An aberrant pattern of DNA methylation in cloned embryo may be abnormal development of fetus, embryonic lethality and placenta dysfunction. The somatic specific band (190kDa) was appeared in ovary and testis, but oocyte specific band (175kDa) was not. Further investigations are necessary to understand the complex links between the methyltransferases and the transcriptional activity of genes in the cloned bovine tissues.

• Journal of Gastric Cancer
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• 제1권3호
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• pp.129-135
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• 2001

Purpose: Dysplasia or flat adenoma of the stomach is regarded as a precancerous lesion. However, the frequency and the evolutionary process of malignant transformation of gastric dysplasia are still debated. In order to see whether the lesion was a monoclonal or a polyclonal proliferation, clonality was assayed by X-linked HUMARA polymorphism. Materials and Methods: DNA was extracted from the paraffin-embedded tissue of 16 consecutive cases of endoscopic biopsy, eight of which supplied both dysplastic and nondysplastic tissue for comparison. HUMARA was amplified by PCR with or without pretreatment with methylationsensitive restriction enzyme, HpaII. The amplification products were electrophoresed on polyacrylamide gel and silver-stained. Results: Among the 16 cases, 13 cases were informative and 3 cases noninformative. Of the 13 cases, one case showed skewed lyonization, rendering 12 cases to be analyzed further. A monoclonal band pattern was noted in 2 cases, and a polyclonal band pattern in 10 cases. A review of the histopathologies of the monoclonal and the polyclonal cases did not reveal features discriminating the two groups. Conclusion: These results suggest that gastric dysplasia is a disease entity heterogeneous in the genetic level, and many cases may be non-neoplastic.

• Asian Pacific Journal of Cancer Prevention
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• 제16권4호
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• pp.1615-1619
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• 2015

Background: Promoter hypermethylation mediated gene silencing of tumor suppressor genes is considered as most frequent mechanism than genetic aberrations such as mutations in the development of cancers. BRD7 is a single bromodomain containing protein that functions as a subunit of SWI/SNF chromatin-remodeling complex to regulate transcription. It also interacts with the well know tumor suppressor protein p53 to trans-activate genes involved in cell cycle arrest. Loss of expression of BRD7 has been observed in breast cancers and nasopharyngeal carcinomas due to promoter hypermethylation. However, the genetic status of BRD7 in oral squamous cell carcinomas (OSCCs) is not known, although OSCC is one of the most common among all reported cancers in the Indian population. Hence, in the present study we investigated OSCC samples to determine the occurrence of hypermethylation in the promoter region of BRD7 and understand its prevalence. Materials and Methods: Genomic DNA extracted from biopsy tissues of twenty three oral squamous cell carcinomas were digested with methylation sensitive HpaII type2 restriction enzyme that recognizes and cuts unmethylated CCGG motifs. The digested DNA samples were amplified with primers flanking the CCGG motifs in promoter region of BRD7 gene. The PCR amplified products were analyzed by agarose gel electrophoresis along with undigested amplification control. Results: Methylation sensitive enzyme technique identified methylation of BRD7 promoter region seventeen out of twenty three (74%) well differentiated oral squamous cell carcinoma samples. Conclusions: The identification of BRD7 promoter hypermethylation in 74% of well differentiated oral squamous cell carcinomas indicates that the methylation dependent silencing of BRD7 gene is a frequent event in carcinogenesis. To the best of our knowledge, the present study is the first to report the occurrence of BRD7and its high prevalence in oral squamous cell carcinomas.

• Pediatric Infection and Vaccine
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• 제12권2호
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• pp.166-177
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• 2005

목 적 : DNA 제한 효소 분석법을 이용한 아데노바이러스의 유전체형 분석 방법은 많은 연구자들이 서로 다른 종류의 제한 효소와 명명법을 사용하여 분류함으로써, 아직까지 체계적인 분류 체계가 정립되어 있지 않다. 본 연구는 Li와 Wadell이 제안한 제한 효소 분석법과 명명법을 이용하여 국내에서 최근 14년 동안 분리된 아데노바이러스 혈청형 1, 2, 5형에 대한 유전체형을 분류하고, 그 분자 역학과 유전체형 상호 간의 연관성을 밝히고자 시행하였다. 방 법 : 1990년 11월부터 2003년 2월까지 하기도 감염으로 서울대학교병원 소아과에 입원하였거나, 인접 지역의 종합병원 소아과에 입원한 소아들로부터 채취한 비인두 흡인물을 검체로 하여 HEp-2 세포주에서 배양 후 간접면역형광검사로 확인하고, 분리된 아데노바이러스를 항혈청 1, 2, 3, 4, 5, 6, 7, 11에 대한 세포독성 효과를 관찰함으로 혈청형을 결정하였다. 혈철형 1, 2, 5형을 대상으로 DNA를 추출하고 12가지 제한 효소 BamHI, BcI, BglI, BglII, BstEII, EcoRI, HindIII, HpaI, SalI, SmaI, XbaI, XhoI로 절단한 후 전기영동 시키고 나타나는 분절 형태를 각 혈청형의 표준주와 비교하여 분석하였다. 유전체형간의 상관성을 비교하기 위하여 PCRF 분석을 시행하였다. 결 과 : 아데노바이러스 분리주 382주를 대상으로 혈청형을 확인한 결과, 1형 33(9%), 2형 45(12%), 3형 107(28%), 4형 16(4%), 5형 24(6%), 6형 8(2%), 7형 116(30%), 11형 9(2%), 그 외의 형들이 24(6%)주로 각각 나타났다. 변이 유전체형은 혈청형 1형 18종류, 2형 25종류, 5형 10종류가 분류되었으며 Ad1p1-Ad1p7, Ad1a, Ad1b, Ad1b1-Ad1b3, Ad1c, Ad1d, Ad1e, Ad1e1, Ad1e2, Ad1f; Ad2p1-Ad2p11, Ad2a, Ad2a1-Ad2a6, Ad2b, Ad2c, Ad2d, Ad2e, Ad2e1-Ad2e3; Ad5p1, Ad5p2, Ad5a, Ad5a1-Ad5a7로 명명되었다. 본 연구에서는 표준주나 이전에 보고 된 유전체형과 일치하는 유전체형은 분리되지 않았다. 대부분의 유전체형은 전 연구기간 동안 1~2주만 분리되었고 일부 유전체형들은 산발적으로 2회 이상 반복 분리되었다. 유전체형 Ad1p5나 Ad5a1와 같이 유행성으로 분포하는 유전체형도 관찰되었다. 혈청형 1형 유전체형 간의 PCRF는 79~99%로 Genomic cluster 1과 2로 구분되었고, 2형과 5형은 각각 82~99%와 84~99%로 모두 80% 이상이었다. 결 론 : 본 연구를 통하여 국내에서 분리된 아데노바이러스 혈청형 1, 2, 5형의 다양한 유전체형을 체계적으로 분석할 수 있었다. 혈청형 1, 2, 5형이 주로 산발성 감염을 일으키는 것으로 알려져 왔으나, 유전체형에 따라 역학적 특징이 다르게 나타날 수 있으며, 이는 DNA의 변형에 의한 유전체형의 변화가 바이러스의 감염력과 생존력에 변화를 일으켰을 가능성을 시사한다. 본 연구 결과는 혈청형 1, 2, 5형의 비교 자료로 활용될 수 있을 뿐만 아니라, 변이 유전체형들에 대한 정보를 제공함으로써 백신 개발을 위한 기초 자료로 활용될 수 있을 것으로 기대된다.