Differences in DNA methylation patterns are detectable during the dimorphic transition of fungi by amplification of restriction polymorphisms

Abstract

A modification of the amplified fragment length polymorphism technique was developed for the determination of DNA methylation in dimorphic fungi 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 isoschizomers MspI and HpaII, whose ability to cleave at the sequence CpCpGpG is affected by the methylation state. The resulting fragments were ligated to primers and subjected to a double round of amplification by the polymerase chain reaction, radiolabeled in the second round, and separated by polyacrylamide gel electrophoresis. Comparison of patterns revealed differences indicative of fragments whose methylation state did or did not change during the dimorphic transition. These results indicate the usefulness of the method for the study of DNA methylation, demonstrate the universality of DNA methylation in fungi, and confirm that differential DNA methylation occurs during fungal morphogenesis.

Genetic and epigenetic variation in Spartina alterniflora following the Deepwater Horizon oil spill

“…We used MS‐AFLP to assess genomewide DNA methylation on the same duplicate DNA extractions used in the AFLP protocol (Reyna‐Lopez, Simpson, & Ruiz‐Herrera, 1997). We used MspI and HpaII restriction enzymes, which have different sensitivities to cytosine methylation of the same CCGG sequence (Reyna‐Lopez et al., 1997; Salmon, Clotault, Jenczewski, Chable, & Manzanares‐Dauleux, 2008).…”

“…We used MspI and HpaII restriction enzymes, which have different sensitivities to cytosine methylation of the same CCGG sequence (Reyna‐Lopez et al., 1997; Salmon, Clotault, Jenczewski, Chable, & Manzanares‐Dauleux, 2008). DNA extracts were digested with both EcoRI/MspI and EcoRI/HpaII enzyme combinations independently for each individual, and selective PCR was run with fluorescently labeled primers EcoRI  + AGC (6‐FAM) and +ACG (HEX) and unlabeled primers HpaII/MspI +TCAC and HpaII/MspI  + TCAT.…”

Detection of epigenetic variation in tissue-culture-derived plants of Doritaenopsis by methylation-sensitive amplification polymorphism (MSAP) analysis

“…The MSAP technique is a relatively new modification of the amplification fragment length polymorphism technique, first developed to determine DNA methylation events in dimorphic fungi (Reina-Lopez et al 1997) and later adopted for the detection of cytosine methylation in various plants including rice (Xiong et al 1999) and banana (Peraza-Echeverria et al 2001;Baurens et al 2003). This technique is useful for the analysis of epigenetic variations in apple (Li et al 2002) and hop (Peredo et al 2006).…”

Analysis of DNA Methylation in Various Swine Tissues

“…The methylation-sensitive amplification polymorphism (MSAP) technique is a relatively new modification of the amplification fragment length polymorphism (AFLP) technique; MSAP was first developed to determine DNA methylation events in dimorphic fungi [20]. The MSAP technique utilizes the restriction isoschizomer pair HpaII and MspI [20] (instead of MseI as in the original protocol [21]).…”