Journal: Molecular and Cellular Biology
Article Title: Cooperativity between DNA Methyltransferases in the Maintenance Methylation of Repetitive Elements
Figure Lengend Snippet: (A) Methylation-sensitive fingerprints of M1/3A/3B, M3A/3B, and M1 ES cell DNA after enzyme digestion with either Rsa I, Rsa I with Hpa II, or Rsa I with Msp I for 16 h. Bands which appear to be hypomethylated only in lane 2 are indicated by solid arrows (class I). Bands which appear to be hypomethylated in both lanes 2 and 3 are indicated by open arrows (class II). Solid squares, functional Dnmt genes; open squares, inactivated genes. GCP1, -2, and -4 are GC-poor primers. (B) Southern blot analysis of genomic DNA from M1/3A/3B, M3A/3B, and M1 ES cells using isolated AP-PCR fragment CI-f as a probe. (C) Ms-SNuPE analysis for fragment CII-d from M1/3A/3B, M3A/3B, and M1 ES cell DNA. Quantitative methylation analysis of three CpG sites in CII-d was performed. The percent methylation given under each gel represents the average obtained for the three sites by using the following equation with PhosphorImager quantitation results: [C/(C + T)] × 100, where C is methylated and T is unmethylated. CpG sites are labeled; hatched boxes, repetitive sequences; arrows, PCR primers.
Article Snippet: Ten micrograms of DNA from cell lines was separately digested with either 50 U of Rsa I, 50 U each of Rsa I and Hpa II, or 50 U each of Rsa I and Msp I (Roche) at 37°C for 16 h. Digested genomic DNA was electrophoresed on a 0.7% agarose gel and Southern transferred to Zeta-Probe (Bio-Rad) membranes overnight.
Techniques: Methylation, Functional Assay, Southern Blot, Isolation, Polymerase Chain Reaction, Mass Spectrometry, Quantitation Assay, Labeling