Examining the Role of Cytosine Methylation in C. cinereus CpG methylation is known to be involved for gene silencing and to be responsible for genomic imprinting. We wish to understand the consequences of cytosine methylation in the fungal system Coprinus cinereus. In this study we identify the location and extent of 5-methyl cytosine residues in the tryptophan synthetase gene (trpl). It was shown previously that cytosine methylation can occur as a result of a duplication of the trpl gene in the C. cinereus genome. And, we make use of methylating enzymes to methylate cytosine residues in the exogenous gene Hygromycin B Phosphotransferase (PHT). We use a genomic sequencing protocol whereby cytosine is deaminated to uracil upon treatment with sodium bisulfite while 5-methyl cytosine remains unchanged. Thus, the presence of 5-methyl cytosine (m5C) residues is indicated by the remaining cytosines in the sequence data. We will analyze the trpl gene sequence in six tetrads that have both Trp+ and Trp- progeny to make a direct comparison of differe nces in methylation between silenced and unsilenced genes. This sequence analysis will determine if there is site specificity for cytosine methylation, and if this methylation occurs exclusively at CpG dinucleotides. In addition, we may learn if specific methylated cytosines are cytosines are necessary and sufficient to render the trpl gene inactive. We will further determine if maintenance methylation exists when an exogenous gene is methylated in vitro and transformed into C. cinereus. 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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