Fig. 2. The spiel ohne grenzen mutations are linked to point mutations in pou2. (A) Genomic structure of the transcribed region of the pou2 locus. The coding region is interrupted by four introns. Translational start site and stop codon are indicated. The POU-specific domain is shown in blue, and the POU-homeodomain is shown in green. (B) Mapping of the spg locus, and demonstration of tight linkage to the pou2 gene. Molecular markers used for meiotic mapping are indicated in blue. PACs isolated in the genomic walk are designated A, B and C (see Materials and Methods). Recombination frequencies of the polymorphic markers used are indicated. The map positions of the markers are from the Stanford HS panel (Kelly et al., 2000), except for Z13467 (six recombinants/168 meioses), Z21718 (2/556), Z12068 (2/1666) and Z7224 (21/556), which are from the MGH panel (Shimoda et al., 1999). Z21718 maps on the HS panel at 33.1 cM. (C) Putative Pou2 protein products generated by spg mutant alleles. Wild-type and mutant proteins are shown at top and bottom, respectively. Foreign sequences, generated by frameshift, are indicated in red. The asterisk shows the position of the mis-sense mutation in helix 1 of the homeodomain in spgm216. The splice acceptor sites in spgm793 and spgm308 are highlighted in gray. (D) Top: sequence comparison of pou2 genomic DNA from wild-type embryos and mutants. Mutated positions are underlined. Bottom: genotype analysis of single embryos. Each spg mutation eliminates a restriction site. As expected, these restriction site polymorphisms segregate with the mutant phenotypes. Genotypes are indicated by ‘+’ (wild type) and ‘-’ (mutant). wt, wild type; un, undigested PCR product.
