Molecular identification of the suf locus.
(A) Diagram of zebrafish oogenesis represented as a composite of wedges displaying a single stage . Oocytes are not drawn to scale and in the ovary, stages are mixed. Ia: 7–20 μm diameter of the oocyte; oocytes are still connected forming oogonia after the last mitosis. Ib: 20–140 μm, oocytes are separated by a layer of follicle cells (dashed red-yellow line) and several nucleoli appear in the nucleus (grey). II: 140–340 μm, formation of cortical granules is initiated (white vesicles). III: 340–690 μm, massive accumulation of yolk globules (brown vesicles) during vitellogenesis covering the germinal vesicle (oocyte nucleus). IV: 690–730 μm, oocytes are transformed into a fertilizable egg by meiotic maturation inducing germinal-vesicle-break-down. Simultaneously, the cytoplasm becomes transparent by yolk protein cleavage. V: 730–750 μm, eggs are released from the follicle layer of the ovary during ovulation into the oviduct. (B) Stage V oocytes 10 min after activation from heterozygous wild-type (wt; left panel) and mutant mothers (suf; right panel) showing the opaque cytoplasm and the chorion elevation defect in the mutant. Scale bar: 100 μm. (C) Genetic map of the suf-locus on chromosome 13. Meiotic mapping located the mutation between the markers z25580/G47633 (0.4 cM, centiMorgan; 5 recombinants/1183 females) and z21403/G41743 (0.4 cM) corresponding to a physical interval of 1.22 Mb (megabases). Fine mapping of the suf-locus identified markers AL13-10 (0.08 cM) and AL13-13 (0.2 cM), which physically represents 280 kB. (D) Among other genes, this interval contains the ArgII, vti1b, rdh12, galectin and pleckstrin2 genes, whose cDNA sequence did not display mutations in comparison to the database genome (http://www.ensembl.org/Danio_rerio/Info/ Index). (E) Exon-intron structure of the suf gene. (F) The p96re allele carries a G-A transition at the genomic level destroying a splice donor site in the transcribed RNA. (G) The selection of an alternative, upstream splice donor leads to a deletion of 25 nucleotides causing a frameshift in the cDNA encoding six aberrant amino acids and eventually creating a premature STOP-codon (asterisk). (H) The mutant protein is predicted to lack 282 amino acids at the C-terminus including the conserved SUF domain from amino acid 2375 to 2552. (I) Phylogenetic diagram displaying the conservation of Souffle proteins among vertebrates. The Anopheles and Drosophila proteins were used to root the tree. Numbers indicate bootstrap-values and the scale the number of substitutions per amino acid residue.