DUPLICATION-DEPENDANT DNA METHYLATION IN C. CINEREUS

胞嘧啶的DNA甲基化是一种已知的现象 真核生物。有人认为，真核细胞的DNA甲基化是 负责基因沉默，更具体地说，负责基因组印记。 这项研究的目的是确定 色氨酸合成酶基因(TRPL)中5-甲基胞嘧啶残基的研究 真菌系统，灰鸡腿菌。胞嘧啶甲基化以一种 灰霉菌基因组中trp1基因复制的结果。在这 研究中，使用基因组测序方法检测到胞嘧啶甲基化 (Frommer，1992)，治疗后胞嘧啶脱氨基为尿嘧啶 使用亚硫酸氢钠，而5-甲基胞嘧啶保持不变。DNA 样本是基于甲基化的检测而选择的 甲基酶敏感限制性内切酶(HPAⅡ)。这些样本显示出 与质粒DNA相比，出现了不同的带型。两种质粒 用钠处理DNA(未甲基化)和子代DNA(甲基化) 亚硫酸氢盐，用链特异性引物进行聚合酶链式反应，克隆到 PCRII载体并测序。5-甲基胞嘧啶的存在 序列数据中任何剩余的胞嘧啶都证实了残基。 对选定克隆的序列分析显示存在胞嘧啶 子代DNA甲基化与对照质粒DNA的甲基化 没有胞嘧啶甲基化。C.cinereus已被证明是一个模型系统 研究甲基化，可能给我们提供关于基因沉默是如何发生的线索 在这个物种中，可能在整个真核生物中。

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.