DIFFERENCES IN DNA METHYLATION PATTERNS ARE DETECTABLE DURING THE DIMORPHIC TRANSITION OF FUNGI BY AMPLIFICATION OF RESTRICTION POLYMORPHISMS

A modification of the amplified fragment length polymorphism technique was developed for the determination of DNA methylation in dimorphic f ungi representative of three of the major fungal taxa: Mucor rouxii, a zygomycete; Yarrowia lipolytica, an ascomycete; and Ustilago maydis, a basidiomycete. DNA obtained from the yeast or mycelial stages of the fungi was digested with a mixture of EcoRI, and one of the isoschizom ers MspI and HpaII, whose ability to cleave at the sequence CpCpGpG is affected by the methylation state. The resulting fragments were ligat ed to primers and subjected to a double round of amplification by the polymerase chain reaction, radiolabeled in the second round, and separ ated by polyacrylamide gel electrophoresis. Comparison of patterns rev ealed differences indicative of fragments whose methylation state did or did not change during the dimorphic transition. These results indic ate the usefulness of the method for the study of DNA methylation, dem onstrate the universality of DNA methylation in fungi, and confirm tha t differential DNA methylation occurs during fungal morphogenesis.