Growth and Methylation Defects in Atrxnull ES Cells
(A) Cultures were inoculated with equivalent numbers of ES cells bearing different Atrx alleles as indicated, and were serially passaged. After the indicated days of coculture, DNA extracted from a sample of cells was analysed by Southern blot to detect the Atrx alleles. DNA was digested with SpeI, and the membrane was hybridised with the 20/27 probe shown in Figure 2A. The expected sizes of the different alleles are indicated.
(B) Schematic diagram of the transcribed portion of the mouse rDNA repeat with the 18S, 5.8S, and 28S genes indicated. The positions of the limit-digesting enzymes BamH (labelled B) and EcoRI (labelled E) and the probes (RIB3 and RIB4) used in the Southern blots shown in (C) are indicated. Below are shown the locations of the methylation-sensitive enzymes (SmaI, PvuI, and MluI) whose methylation status has been analysed in the Southern blots shown in (C).
(C) DNA from Atrx-positive (Atrx+, bearing either an Atrx WT or Atrx flox allele) or Atrxnull (bearing the Atrx Δ18Δneo allele) ES cells and 7-d embryoid bodies were digested with the enzymes shown and analysed by Southern blotting using the probes indicated. Arrows indicate the fully methylated copies (cut by only the limit-digesting enzyme). Phosphorimager quantitation of the blots are shown below. The y-axis shows the percentage of copies that are undigested by the methylation-sensitive enzyme as a percentage of the total signal from cut and uncut rDNA. Mean values are indicated by horizontal lines, and the significance of the differences between the Atrx-positive and Atrxnull populations are shown for each enzyme.