DUPLICATION-DEPENDANT DNA METHYLATION IN C. CINEREUS

C. Cinereus 中重复依赖性 DNA 甲基化

• 批准号: 5210034
• 负责人: SYLVIA A FRAZIER-BOWERS
• 金额: --
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/5210034
• 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.

胞嘧啶的DNA甲基化是一种已知的现象， 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，基因组印记。 本研究的目的是确定的位置和范围 5-色氨酸合成酶基因（trpl）中的甲基胞嘧啶残基， 真菌系统，灰盖鬼伞。 胞嘧啶甲基化发生在 trp 1基因在C.灰蝶基因组 在这 在一项研究中，使用基因组测序方法检测胞嘧啶甲基化 （Frommer，1992）治疗后，胞嘧啶脱氨为尿嘧啶 用亚硫酸氢钠，而5-甲基胞嘧啶保持不变。 的DNA 基于使用甲基化的检测选择样品。 甲基化酶敏感限制性内切酶（Hpa II）。 这些样本显示， 与质粒DNA相比具有不同的带型。 两种质粒 DNA（未甲基化的）和子代DNA（甲基化的）用钠处理， 亚硫酸氢盐，使用链特异性引物用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。 所选克隆的序列分析显示胞嘧啶的存在 子代DNA中的甲基化与对照质粒DNA相比， 没有胞嘧啶甲基化。 C. cinereus已经被证明是一个模型系统， 研究甲基化，可能会给我们提供基因沉默发生的线索， 在这个物种中，可能在整个真核生物中。