Fig. 5. Identification of protein 4.1R as the mutated gene in mot/cha. (A) cDNA sequence comparison of wild-type and both alleles of mot shows two nonsense point mutations in the mot alleles (point mutations are underlined). The DNA sequence of mot^tm303c and its corresponding wild-type sequence shown here are in the 3'to 5' orientation. (B) A protein truncation test shows the truncated translation product of mot^tu275 cDNA compared with that of wild-type cDNA. (C) Linkage analysis via allele specific PCR primers on genomic DNA from wild-type (lanes 1-4), heterozygous (lanes 5-8) and mot (lanes 9-12). The wild-type primer, top, amplifies wild-type and heterozygous DNA, whereas the mutant primer, bottom, amplifies DNA from heterozygous and mot embryos. (D) Genetic map of the cha locus on LG16. EST Fb70c02 showed no genetic recombinants from the cha locus in 127 informative animals. (E) Allele-specific oligonucleotide hybridization for wild-type and mutant protein 4.1R sequences. Wild-type siblings (lanes 1-7) and mutant siblings (lanes 8-14) show complete linkage with either normal or mutant protein 4.1R alleles, respectively. (F) P4.1R in situ hybridization shows an expression pattern restricted to the hematopoietic intermediate cell mass. Wild-type 24 hpf embryos exhibit high-level expression of the P4.1R transcript, whereas the level of transcript expression is greatly reduced in mot embryos. (G) At 96 hpf, mot embryos that were injected with P4.1R/GFP construct are partially rescued and have a higher number of circulating red cells compared with mot controls.