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甲基化是一种已知现象 真核生物。 有人提出真核DNA甲基化是 负责基因沉默，更具体地说，基因组印记。 这项研究的目的是确定 色氨酸合成酶基因（TRPL）中的5-甲基胞嘧啶残基使用 真菌系统，cinereus。 胞嘧啶甲基化作为一种 C. cinereus基因组中TRP1基因重复的结果。 在这个 研究，使用基因组测序方法检测到胞嘧啶甲基化 （Frommer，1992）在治疗时胞嘧啶脱氧于尿嘧啶 甲基硫酸钠，而5-甲基胞嘧啶保持不变。 DNA 根据使用A检测甲基化选择样品 甲基酶敏感限制酶（HPA LL）。 这些样品显示 与质粒DNA相比，不同的带模式。 两个质粒 用钠处理DNA（未甲基化）和后代DNA（甲基化） 用链特异性引物用PCR扩增的硫酸盐，克隆到 PCRII载体并测序。 5-甲基胞嘧啶的存在 序列数据中的任何剩余细胞菌都证实了残基。 选定克隆的序列分析揭示了胞嘧啶的存在 后代dnvers中的甲基化对照质粒DNA，揭示了 无胞嘧啶甲基化。 C. Cinereus已被证明是 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，可能在整个真核生物中。