Fig. 5. Identification and characterisation of the Mnt mutation. (A) Representation of three cosmids used to screen for alterations in the Igf2-H19 region. The approximate location of the Mnt mutation is marked with #. (B) Hybridisation of cosmid CH to homozygous (Mnt) and wild-type EcoRI and BamHI digested genomic DNA. Note differences in bands between Mnt and wild-type samples are marked with *. (C) Arrangement of the two breakpoint regions on a wild-type chromosome. The breakpoint 1 (BP1) region (downstream of H19) is shown as a red line, while the BP2 region which is 3.5 cM further centromeric is shown as a blue line. The regions conserved between mouse and human (1-10) as identified by Ishihara et al. (Ishihara et al., 2000) are shown (green squares), while the region (+22 to +28 kb from the H19 promoter) identified by Kaffer et al. (Kaffer et al., 2000) as possessing enhancer activity is shown by a black line. (D) The arrangement of the two breakpoint regions on the Mnt chromosome. Note that the rearrangement isolates the conserved elements 9 and 10 from the H19 region. PCR primers (1-4) for the detection of the breakpoints are shown. (E) Relative positions of the two Mnt breakpoints, the surrounding genes and the genetic distance between the two breakpoints. The red and green lines represent the probes used in the FISH analysis. (F) Fluorescence in situ hybridisation (FISH) analysis on heterozygous Mnt/F1 nuclei, verifying that an inversion has occurred on the Mnt chromosome. Red, H19 probe; green, BP2 probe. In the merged image, 1 is the H19 region on the wild-type chromosome; 2 is the BP2 region on the wild-type chromosome; 3 is the 3' part of the BP2 region on the Mnt chromosome (see E); and 4 is the co-localisation of the 5' part of the BP2 region and the H19 region on the Mnt chromosome (yellow).