Fig. 2. jef^tw37 alters a conserved splice site sequence in sox9a and inhibits message splicing. (A) The sequence of sox9a in zebrafish wild-type strains AB and TÜ, splice junction (arrow). An in frame stop codon directly followed the G to T change in jef^tw37. (B) The primer pair f1/r1 in exon 1 and intron 1 amplified a band of the expected size (544 bp) from wild-type (WT) genomic DNA and from jef^tw37 cDNA, showing that intronic sequences of sox9a were present in mutant cDNA. With normal splicing in wild-type cDNA, there was no strong product of this size. (C) RNase protection assays (RPA, upper panel) on RNA extracted from wild-type and homozygous jef^tw37 mutants 4 days old. tRNA served as a negative control. odc, the internal control. Probe for RPA is made from a 402 bp of sox9a cDNA amplified using primer pair f2/r2. Quantifying band intensity (lower panel) shows a drastic decrease of message in jef^tw37 embryos. (D,E) The jef^tw37 allele behaves as a null when heterozygous for the deletion allele b380. bh, basihyal; bsr, branchiostegal rays; cb, ceratobranchials; ch, ceratohyal; ch?, presumed ceratohyal; cl, cleithrum; ed, endoskeletal disk; co, scapulocoracoid; hs, hyosymplectic; m, Meckel's cartilage; m/de? putative Meckel's cartilage and dentary bone; op, opercule; pq, palatoquadrate. Scale bar: 100 µm.