Positional cloning of the miak mutation.

A. Genetic maps obtained by genotyping and phenotyping of the progeny from the intercross between (C57BL/6J-miak/miak congenic mice × C57BL/6J) F2. The blue markers D19Mit 112 and D19Mit74 define the non-recombinant interval containing miak mutation and Pitx3 that is responsible gene for the mouse Pitx3ak and Pitx3eyl mutation [5], [9]. Distances on chromosome 19 are according to the mouse mm 10 (Genome Reference Consortium GRCm38) genomic sequence. B. Mutation analysis of Pitx3 in the miak mouse. miak mice have a c.444C>A nonsense mutation in Pitx3. C. Schematic diagram of the domain structure of the PITX3 protein in the +/+ and miak mice. The domain structures were predicted by the SMART program, and the numbering of the amino acids (aa) is according to the PITX3 aa sequence of the wild-type and miak mice (NP_032878 and AB971349). PITX3 possesses homeodomain (HD, black box) and otp, aristaless, and rax (OAR, light gray box) as major functional domains near the N- and C-termini, respectively. The nonsense mutation in the miak mutants cause truncations of the PITX3 protein that result in a missing C-terminal OAR domain. D. The miak mutation disrupts a SmaI restriction site (CCCGGG) in Pitx3. The digestion of amplicons from wild-type mice produces bands at 136 and 132 bp. However, miak/miak mice are homozygous for the disruption of the SmaI site and yield only a single 268 bp band, whereas the miak/+ mice are heterozygous for the mutation as shown by the two banding patterns superimposed on one another. The top and bottom panels show the RFLP patterns of (C57BL/6J-miak/miak congenic mice × C57BL/6J) F2 progeny and wild-type inbred strains, respectively. M, marker (100 bp ladder); N, negative control (DDW); CIS, common inbred strain; Dom, domesticus; Mol, molossinus; Mus, musculus; Cas, castaneus.