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.
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