Fig. 4

A premature stop codon in spermidine synthase is responsible for the idefix phenotype. (A) The ide mutation, t26743, was placed between the two z-markers z3199 and z14967 by mapping of meiotic recombinants. The region contains 14 annotated protein coding genes and two microRNA genes. (B) ide^t26743mutants carry a mutation in exon 2 of srm. The gene structure is shown in the top panel, the bottom panel shows the sequence chromatograms of wild-type and homozygous mutant fish. The mutated nucleotide is marked with an asterisk. (C) An alignment of the amino acid sequences of spermidine synthases from zebrafish (Danio rerio), humans (Homo sapiens) and E.coli. Positions of conserved amino acid residues important for substrate binding and catalytic activity are highlighted in red. The position of the mutation in ide^t26743 (Leu53 to Stop) is underlaid in red. (D) An F₀ fish is shown, which was obtained after CRISPR-mediated knockout of srm. (E) The sequence chromatograms from the fish in D. The induced mutations lead to a deterioration of the signal quality in both directions at the same position. (F) A homozygous mutant F₂ fish carries an 80bp deletion and shows the typical ide mutant phenotype. (G) The biosynthetic pathway for the generation of the polyamines putrescine, spermidine and spermine from ornithine and S-adenosyl-methionin-amine. Scale bars: 5mm.