A TARGETED-REPLACEMENT SYSTEM FOR IDENTIFICATION OF SIGNALS FOR DE-NOVO METHYLATION IN NEUROSPORA-CRASSA

Transformation of eukaryotic cells can be used to test potential signa ls for DNA methylation. This approach is not always reliable, however, because of chromosomal position effects and because integration of mu ltiple and/or rearranged copies of transforming DNA can influence DNA methylation. We developed a robust system to evaluate the potential of DNA fragments to function as signals for de novo methylation in Neuro spora crassa. The requirements of the system were (i) a location in th e N. crassa genome that becomes methylated only in the presence of a b ona fide methylation signal and (ii) an efficient gene replacement pro tocol. We report here that the am locus fulfills these requirements, a nd we demonstrate its utility with the identification of a 2.7-kb frag ment from the psi(63) locus as a new portable signal for de novo methy lation.