Fig. 4
The tangohkz5 mutant phenotype is caused by a nonsense mutation in sae1. (A) The tangohkz5 mutation was mapped on chromosome 15 between the SNP marker zgc154093 (two recombinants out of 1200 tangohkz5/hkz5 embryos) and zk31M14-204059 (four recombinants out of 1200 tangohkz5/hkz5 embryos). This region contains four genes: sae1, LOC557823, bbc3 and zgc154093. (B) The sae1 gene contains nine exons and a C-to-T substitution was found in exon 7. (C) Western blotting of 293T cells transfected with pCS2+-Flag-wt sae1 or pCS2+-Flag-mut Δsae1. The expression level of the mutant ΔSae1 protein was much lower than that of wild-type Sae1. Tubulin was used as a loading control. Real-time PCR showed the similar RNA levels of wild-type and mutant forms of sae1 relative to Gapdh (P=0.3, n=3). (D) Co-immunoprecipitation assay. 293T cells were co-transfected with either pCS2+-Flag-wt sae1/pCS2+-Myc-sae2, or fivefold pCS2+-Flag-mut Δsae1/pCS2+-Myc-sae2. Transfection with pCS2+-Flag-wt sae1/pCS2+-Myc was used as a negative control. The input showed the similar expression levels of either Flag-Sae1 or Myc-Sae2 between wild type and mutant. Quantification showed that the co-immunoprecipitated mutant ΔSae1 was decreased by >50% compared with wild-type Sae1. The data were reproducible in two independent experiments. (E-H) WISH shows the cmyb expression in the CHT of wild-type siblings (E), tangohkz5/hkz5 embryos (F), tangohkz5/hkz5 embryos injected with sae1 wild-type mRNA (G), and tangohkz5/hkz5 embryos injected with the mutant Δsae1 mRNA (H). Error bars represent s.e.m. |