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DAF-9, a cytochrome P450 regulating C. elegans larval development and adult longevity

Molecular Biology Program and Division of Biological Sciences, University of Missouri, Columbia, MO 65211, USA

View larger version (129K): [in a new window]   Fig. 1. daf-9 mutant phenotype. (A) C. elegans hermaphrodite reproductive system. The gonad consists of anterior and posterior U-shaped tubular arms, with ovaries connecting to ventral spermathecae, which then join to the uterus, terminating at the vulva. (B) Normal morphology in a daf-9(e1406) daf-12(m20) double mutant. (C) Abnormal reproductive system of a daf-9(e1406) adult. Vesicles are present in much of the space normally occupied by the uterus. A large pseudovulval protrusion is shown. (D) Abnormal distal tip cell migration in daf-9(e1406). The anterior gonad arm did not reflex dorsally, but extended anteriorly to the pharynx. (E) Molting defect of daf-9(e1406). A shed anterior cuticle remains attached to the body, resulting in a constriction. rg, dorsal ovary of the reflexed gonad; vul, vulva; pv, pseudovulval protrusion; dtc, distal tip cell; v, vesicle; ac, shed anterior cuticle; *, constriction. Scale bars: 10 µm.

View larger version (30K): [in a new window]   Fig. 2. Survival of wild type (N2), daf-9 mutants and daf-d; daf-9 double mutants. Each curve depicts one of the two independent trials represented in Table 1. The life spans were determined from the percentage of worms alive on a given day. (A) daf-9 single mutants at three temperatures. (B) daf-d; daf-9 double mutants at 15°C.

View larger version (31K): [in a new window]   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.

View larger version (19K): [in a new window]   Fig. 4. The Daf-9 phenotype is sterol dependent. (A) DAF-9 is required for normal non-dauer development. Recovery of daf-9(m540) from dauer-like arrest was assayed at 20°C on NG agar plates with cholesterol concentrations as shown. Data are presented as percent recovery on a given day after dauer-like arrest. (B) Cholesterol limitation had no effect on the recovery of the daf-7(e1372) mutant, which is Daf-c due to reduced TGF-ß signaling. (C) 7-dehydrocholesterol substituted for cholesterol to promote the recovery of daf-9(m540).

View larger version (81K): [in a new window]   Fig. 5. Expression of daf-9. daf-9p::daf-9cDNA::gfp expression in the daf-9(m540) background. An intense reporter signal was confined to two cell bodies that are posterior to the metacorpus of the pharynx and anterior to the nerve ring. These two cells (arrows) were identified as right and left ventral IL1 neurons, or possibly ventral URA neurons (ventral view). Scale bar, 20 µm.

View larger version (11K): [in a new window]   Fig. 6. Revised pathway for dauer formation and adult longevity. Proposed wild-type function is shown, with arrows indicating stimulation of activities and T-bars indicating inhibition, but the steps do not indicate direct protein interactions. "ON" or "OFF" activity states are given for conditions that promote dauer formation and adult longevity. daf-2 and daf-7 branches converge at daf-9. DAF-9 inhibits dauer formation and shortens life span by inactivating the DAF-12 nuclear receptor (NR). See Discussion for details.

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