Examining the Role of Cytosine Methylation in Coprinus cinereus CpG methylation is known to be involved in gene silencing and to be responsible for genomic imprinting and X-chromosome inactivation. This work addresses the consequences of cytosine methylation in the fungal system Coprinus cinereus and currently is studying the location and extent of 5-methyl cytosine (m5C) residues in the tryptophan synthetase gene (trpi). The genomic sequencing protocol has been refined to study methylation in our fungal system. In the critical reaction of this protocol, cytosine is deaminated to uracil upon treatment with sodium bisulfite while m5C remains unchanged. Thus, the presence of m5C residues is indicated by the remaining cytosines in the sequence data. Analysis of six tetrads with both Trp+ and Trp- progeny allowed me to make a direct comparison of differences in methylation between silenced and unsilenced genes. Sequence analysis of all DNAs proved that cytosine methylation occurs mostly at the CpG dinucleotide as in mammalian systems, and that Trp- DNA has a larger amount of methylated cytosines as compared to Trp+ DNA. Further analysis will determine if there is site specificity for cytosine methylation and if specific methylated cytosines are necessary and sufficient to render the trp1 gene inactive. 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. This may lead to a better understanding of the role of m5C in controlling gene expression and further elucidate its role in diseases such as cancer and the Fragile-X syndrome.
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