Fig. 3. daf-9 encodes a cytochrome P450 hydroxylase. (A) Physical map of the daf-9 region. daf-9 (T13C5.1) is on cosmid T13C5. (B) Three forms of daf-9 cDNA. Lines represent introns and boxes are exons. The predicted steroid-, oxygen- and heme-binding domains of DAF-9 are indicated. Sequence alterations for the five mutations indicated by arrows are described in Results. (C) Alignment of the three important functional domains of DAF-9 with its closest homologs: C. elegans CYP23A1, human CYP17, human CYP21 and Drosophila CYP18. Identical amino acids are in black boxes. The black dot in the oxygen-binding domain indicates the Tc1 insertion sites in m641 and m642 (inserted between the first and second bases in the codon for M356); the arrows indicate two amino acids important for oxygen binding, G360 and T363 (especially T363). The cysteine in the heme-binding domain, C496 (star), provides the proximal thiolate ligand for the heme. (D) Phylogenetic tree showing the relationship of DAF-9 and selected homologs (Nelson, 1998). In addition to the specific gene family members described in the text, rabbit CYP2C1 is included as a representative of the CYP2 family, bovine CYP11A1 represents the mitochondrial (MITO) clan, and wheat CYP51 represents the 51 clan. The GenBank accession number of the daf-9 cDNA sequence is AF407572.