Fig. 2. Molecular analysis of the genomic Rx3 region. (A) CAPS analysis of mutant and wild-type genomic DNA. A 190 bp fragment in HaeIII digested genomic PCR fragment is diagnostic for the mutant Cab allele (el/el and el/+ lanes); the additional restriction site in wild-type Kaga results in 100 bp and 90 bp fragments (arrowhead, el/+ and +/+ lanes). (B) Genomic Southern blot hybridisation using a 600 bp HindIII cDNA fragment comprising exon 1 and 2 (BamHI digest) and a 3 kb genomic DNA fragment (broken line in D; XbaI digest) as probes. In DNA of homozygous mutant embryos (el/el) a larger fragment is detected than in the wild type (+/+), indicating an insertion larger than 13 kb in the mutant el locus. In DNA of heterozygotes (el/+), both fragments are detected. (C) Genomic Southern blot hybridisation with a 4 kb insertion fragment reveals multiple copies in the genome, indicative of middle repetitive DNA. (D) Map of Rx3 region indicating relative distance of B and el on linkage group 12 (LG 12). Red boxes represent three exons on Rx3 cosmid; position of the insertion in intron 2 is shown. Regions comprised in different rescue plasmids and position of the frameshift (fs) are indicated. (E) Predicted amino acid sequence of the Rx3 protein. Splice sites are indicated (arrowheads). Conserved octapeptide (yellow), homeodomain (green) and C-terminal region (red) are highlighted. Abbreviations: B, BamHI; Be, BspEI; S, SnaBI; X, XbaI.