DUPLICATION-DEPENDANT DNA METHYLATION IN C. CINEREUS

C. Cinereus 中重复依赖性 DNA 甲基化

• 批准号: 6238320
• 负责人: SYLVIA A FRAZIER-BOWERS
• 金额: $ 2.26万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-07-01 至 1998-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6238320
• 关键词: 5 methylcytosine; DNA methylation; fungal genetics; gene expression; nucleic acid sequence

DNA methylation of cytosine is a known phenomenon in eukaryotes. It has been suggested that eukaryotic DNA methylation is responsible for gene silencing and more specifically, genomic imprinting. The purpose of this study is to identify the location and extent of 5-methyl cytosine residues in the tryptophan synthetase gene (trpl) using the fungal system, Coprinus cinereus. Cytosine methylation occurs as a result of duplication of the trp1 gene in the C. cinereus genome. In this study, cytosine methylation was detected using a genomic sequencing method (Frommer, 1992) whereby cytosine is deaminated to uracil upon treatment with sodium bisulfite, while 5-methyl cytosine remains unchanged. The DNA samples were selected based on the detection of methylation using a methylase sensitive restriction enzyme (Hpa ll). These samples showed a different banding pattern when compared to the plasmid DNA. Both plasmid DNA (unmethylated) and progeny DNA (methylated) were treated with sodium bisulfite, amplified with PCR using strand-specific primers, cloned into the pCRII vector and sequenced. The presence of 5-methyl cytosine residues was confirmed by any remaining cytosines in the sequence data. Sequence analysis of selected clones revealed the presence of cytosine methylation in the progeny DNversus the control plasmid DNA which revealed no cytosine methylation. C. cinereus has proven to be a model system for studying methylation and may give us clues about how gene silencing occurs in this species and possibly in eukaryotes as a whole.

DNA 胞嘧啶甲基化是已知的现象 真核生物。 有人认为，真核生物 DNA 甲基化是 负责基因沉默，更具体地说，负责基因组印记。 本研究的目的是确定该区域的位置和范围 使用色氨酸合成酶基因 (trpl) 中的 5-甲基胞嘧啶残基 真菌系统，灰盖鬼伞。 胞嘧啶甲基化发生为 C. cinereus 基因组中 trp1 基因复制的结果。 在这个 研究，使用基因组测序方法检测胞嘧啶甲基化 (Frommer, 1992) 治疗后胞嘧啶脱氨基为尿嘧啶 与亚硫酸氢钠反应，而5-甲基胞嘧啶保持不变。 脱氧核糖核酸 使用甲基化检测来选择样品 甲基化酶敏感的限制性内切酶（Hpa II）。 这些样品显示出 与质粒 DNA 相比，带型不同。 两种质粒 DNA（未甲基化）和子代 DNA（甲基化）用钠处理 亚硫酸氢盐，使用链特异性引物通过 PCR 扩增，克隆到 pCRII 载体并测序。 5-甲基胞嘧啶的存在 通过序列数据中任何剩余的胞嘧啶来确认残基。 选定克隆的序列分析揭示了胞嘧啶的存在 子代 DN 与对照质粒 DNA 的甲基化结果表明 无胞嘧啶甲基化。 C. cinereus 已被证明是一个模型系统 研究甲基化并可能为我们提供有关基因沉默如何发生的线索 在这个物种中，也可能在整个真核生物中。