Identifying Sites of Duplication-Dependant DNA Methylation in C cinereus 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. I am using the fungal system, Coprinus cinereus, to identify the location and the extent of 5-methyl cytosine residues in the trpl gene. Cytosine methylation occurs as a result of a duplication of the trpl gene in the C. cinereus genome In this study, cytosine methylation can be detected using a genomic sequencing method (Frommer, 1992) whereby cytosine is deaminated to uracil upon treatment with sodium bisulfite, while 5-methylcytosine remains unchanged. The DNA samples were selected based on the detection of methylation using a methylase sensitive restriction enzyme (Hpa II). These samples showed a different banding pattern when compared to the plasmid DNA. Both plasmid (unmethylated) and progeny (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 is confirmed by any remaining cytosines in the sequence data. Analysis of the sequencing data reveals that there are differences in the extent and location of methylation of cytosine residues. Thus C. cinereus has proven to be a model system for studying methylation and thus may give us clues about how gene silencing occurs in this species and possibly in eukaryotes as a whole.
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