Stanford University

The effect of 5-azacytidine treatment on Mutator activity when applied to developing kernels
--Taylor, R and Walbot, V

MuDr is an autonomous element of the Mutator family inserted into the Bronze2 gene (Bz2). Methylation has been implicated in the down regulation of Mutator activity. To reactivate the inactive Mutator element, developing kernels were treated with 5-azacytidine, an inhibitor of DNA methylation. Mutator stocks containing bz2::MuDr have shown no Bz2 function when crossed to bz2 lines for 6 generations. The reporter line generates excision spots when crossed with an active Mutator line, but when self-crossed or outcrossed to bz2 this inactive line yields only about one excision spot per 50 ears. To assess the role of DNA methylation in the inactivity of MuDr, 5-azacytidine was applied to young ears and excision of the Mutator was observed as anthocyanin spots on mature ears. Using a method previously described (Walbot et al. Maydica 39:19-28, 1994) the husk tissues were carefully peeled back from ears 10-15 days after fertilization and small paper towels soaked in either water or 10 mM 5-azacytidine were applied. The husk tissues were replaced with the aid of an elastic band. Only one application was applied to the ears. The ears were harvested and scored for the presence of excision sectors on the kernels. No excision events were observed in either the water control (6 ears) or the 5-azacytidine treatments (16 ears), suggesting that 5-azacytidine treatment during kernel development does not affect the activity status of inactive MuDr. The experiment will be repeated with additional applications of 5-azacytidine because short exposure time or rapid breakdown of the 5-azacytidine may account for the lack of reactivation. 

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