RNA-directed DNA methylation in Arabidopsis - PubMed (original) (raw)

Structures and methylation analysis of the silencer NOSpro IR (Upper) and target NOSpro–NPTII gene (Lower). NOSpro sequences are depicted as heavy black arrows. Enzymes and probes used for DNA blot analyses are indicated. Abbreviations: Pv, _Pvu_II; D, _Dde_I; S, _Sac_II; N, _Nhe_I; Hi, _Hin_dIII; E, _Eco_RI; B, _Bst_UI; Ba, Bam_HI; P, Pst_I. To assess methylation in the target NOSpro, an E and P double digest was performed (the minus lanes in Figs. 4_A and 6 A, C, E, and G) and one of several methylation-sensitive enzymes (B, D, S, N, Ba) was added. Methylation in the NOSpro IR was tested by digesting with Pv and Hi (the minus lanes in Figs. 4_B and 6 B, D, F, and H), together with either B, D, S, or N. Filled, half-filled, and open circles, squares, and triangles (CG, CNG, and CNN, respectively) indicate >90%, ≈50%, and <10% cytosine (C) methylation, respectively. Open squares or triangles below each map for the enzymes D, N, and B indicate that the top and bottom DNA strands contain C residues in different sequence contexts (e.g., CG and CNG, or CNG and CNN). The _Nhe_I site (underlined) is in the sequence context: 5′-CA

GCTACGmC

AA-3′ (top); and 3′-GTm

CGATCG

TT-5′ (bottom).