Fig. 6

The sec13^sq198 mutation creates a novel splice receptor site in intron7 of sec13. (A) Diagram summarizing map-based cloning of the sec13^sq198 mutant gene. Genetic markers used in mapping are shown on top with their corresponding number of recombinants shown below in bracket. The mutant gene (mu) was located between markers 257H and 257E spanning a 54.3 kb genomic DNA fragment contained in two overlapping BACs (CH211-165I22 and CH211-257F16). (B,C) Analysis of sec13 genomic DNA sequence in sq198 identified a T to A substitution (indicated by red arrows in B) that creates a novel splice receptor site (underlined in red in C, panel for Genomic DNA sequence) in intron 7 of sec13. Native splice receptor site for Intron7/Exon8: underlined in blue. Intron sequence: black letters; exon sequence: red letters. An eight nucleotides insertion (black letters) was identified in the sec13 cDNA derived from sec13^sq198 (C, panel for cDNA sequence). (D,E) Injection of the sec13 splicing blocking morpholino sp2 altered sec13 transcripts with 66 nucleotides deletion (D) that led to the small liver phenotype as conferred by the sec13^m198 mutation when examined with the liver marker fabp10a at 3 dpf (E). (F) Northern analysis of sec13 transcripts in unfertilized eggs (Mt) and 1dpf to 5 dpf embryos using a sec13 probe. 18S RNA was used as loading control. (G) WISH analysis of sec13 expression patterns in unfertilized eggs (Mt), zygotic eggs at 1-cell, 2-cell and 4-cell stages, and in embryos at 3 dpf and 4 dpf. bc: branchial arch; in: intestine; lv: liver; pa: pancreas. (H) Western analysis of Sec13 and Sec31a protein expression, respectively, in unfertilized eggs (Mt) and 1 dpf to 5 dpf embryos. Tubulin was used as loading control.