Figure 4.

Non-CG methylation influences MOM1 transcription. (A) A schematic representation of the genomic region surrounding the MORPHEUS' MOLECULE 1 (MOM1) gene transcription start site (hooked arrow). Recognition sites for the restriction enzymes are indicated with their position relative to the outermost NsiI site. The region used as a probe in (B) is shown. N, NsiI; H, HpaII; S, ScrFI. (B) DNA methylation analysis of the promoter region of MOM1. Genomic DNA of the indicated genotypes was digested with NsiI (methylation insensitive) followed by a secondary digestion with methylation-sensitive restriction endonucleases. The DNA gel blot was probed with a region surrounding the MOM1 transcription start site. MspI and ScrFI are sensitive to non-CG methylation, whereas HpaII is sensitive to both CG and non-CG methylation. The position of size markers is indicated on the right. (C) Transcriptional analysis of MOM1 by semiquantitative reverse transcription–PCR. Negative controls lacked reverse transcriptase (no RT). The size of the amplicons is indicated on the right. Expression of ACTIN 2 was used to normalize the amounts of RNA template. (D) Northern blot analysis of the MOM1 transcript. The same blot was hybridized with a RAN probe as a loading control (bottom). Quantifications of the MOM1 transcript signal were performed on a phosphorimager (Molecular Imager FX; Bio-Rad, Hercules, CA, USA) and are indicated below the RNA blot. Ler, Landsberg erecta; WT, wild type; Zh, Zürich.