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The Caenorhabditis elegans polarity gene ooc-5 encodes a Torsin-related protein of the AAA ATPase superfamily

Section of Molecular and Cellular Biology, University of California, Davis, CA 95616, USA

View larger version (27K): [in a new window]   Fig. 1. Molecular cloning of the ooc-5 locus. (A) Schematic diagram of the genetic and physical maps in the ooc-5 region. The top line shows the rol-6 unc-4 interval of the genetic map of chromosome II (LGII). Below it are shown deficiencies, with the region deleted indicated by hatched bars and the uncertainty in breakpoint shown by broken bars. The physical map of overlapping cosmids (black lines) is shown from left to right: F07H5, T14D7, T07D4, B0457, T21B10, C18E9, F44G4, F37B12, T24B8, C07H4, T08E2. (B) Transformation rescue of ooc-5 mutants. The name of the cosmid or subfragment tested (construct) for rescue is shown on the left, with a plus indicating rescue and the number of lines rescued/total tested shown in parentheses. The extent of each construct is shown on the right. (C) Predicted open reading frames (ORFs) and cDNAs corresponding to the two smallest rescuing fragments. The predicted exons (white boxes) and introns for F44G4.1 (predicted by the C. elegans Genome Project) are shown, with the corresponding regions of identity with cDNAs indicated below. The available sequence tags of yk643a1 and yk452e10 (gray boxes; Wormbase) were used to align these cDNAs, while the SL1 5' clone, a 3' clone and yk504f1 (cross-hatched boxes) were sequenced in their entirety. The 3' cDNA clone includes 168 nucleotides of predicted intron 4. The yk504f1 sequence has 172 nucleotides of predicted intron 4 and a stretch of A residues; this is followed by sequences with identity to T23E1.2, an unrelated ORF, which suggests it is a hybrid cDNA. (D) Schematic representation of OOC-5 domains and homology with other Torsin family members. Black boxes represent predicted signal peptides and gray boxes are additional hyrdrophobic regions. The hatched regions indicate the AAA+ ATPase domain, with the position of the Walker A (A) and Walker B (B) motifs labeled. The larger isoform of OOC-5 is shown; the smaller isoform is missing amino acids 27-32.The amino acid changes associated with the three ooc-5 mutations are indicated by arrows. Percent identity/similarity between OOC-5 and other torsin family members is indicated to the right of each protein; the regions of similarity begin after the hydrophobic domain (amino acid 49 of OOC-5) and continue for the length of the entire proteins. The GenBank Accession Numbers for these sequences are: OOC-5, Z70034 (Accession Number is for cosmid C18E9; the large OOC-5 isoform is C18E9.11b, the short isoform is C18E9.11a); Torsin A, NM_000113; Torsin B, AF317129; Y37A1B.12, CAA19495; and Y37A1B.13, CAA19484.

View larger version (53K): [in a new window]   Fig. 2. Western blot analysis of OOC-5 protein. Blot of whole worm extracts from gravid adult wild-type worms (lane 1) or ooc-5 (it145) homozygous mutant worms (lane 2) probed with anti-OOC-5 antibodies. The arrow indicates the expected position of the OOC-5 protein in the mutant lane. Faint higher molecular weight bands seen in both the wild-type and ooc-5 lanes were also seen in pre-immune controls (not shown). The same blot was probed with anti- tubulin antibodies as a loading control.

View larger version (110K): [in a new window]   Fig. 3. Distribution of OOC-5 in embryos. Confocal micrographs of wild-type embryos (A-O) double labeled with anti-OOC-5 (A,D,G,J,M) and anti-tubulin antibodies (B,E,H,K,N); merged images are shown in C,F,I,L,O, where the overlap in signal appears yellow. Confocal images of ooc-5(it145) mutant embryos double labeled with anti-OOC-5 (P,R) and anti-tubulin (Q,S) antibodies. (A-C) One-cell embryo at prophase; (D-F) one-cell anaphase; (G-I) two-cell interphase; (J-L) two-cell embryo in which the AB cell (left) is in anaphase and P1 is in metaphase. (M-O) Four-cell embryo in which the AB cells are dividing (top left cells, spindle poles only visible), EMS is in prophase and P2 (right most cell) is in interphase. (P-Q) One-cell anaphase ooc-5 mutant embryo. (R,S) Two-cell interphase ooc-5 mutant embryo. The stage of the cell cycle was determined by DAPI staining (not shown) in addition to microtubule staining for all embryos. Posterior is towards the right in this and all figures. Scale bar: 10 µm.

View larger version (85K): [in a new window]   Fig. 4. Comparison of OOC-5, OOC-3 and HDEL staining in wild-type embryos. Confocal micrographs of wild-type embryos double labeled with anti-OOC-5 (A,D,G,J) or anti-OOC-3 (M,P,S) and anti-HDEL antibodies (B,E,H,K,N,Q,T). Merged images showing the extent of signal overlap (yellow) are shown in C,F,I,L,O,R,U. (A-L) Co-localization of OOC-5 and HDEL. (A-C) One-cell prophase embryo with one pronucleus visible; the other pronucleus is out of the plane of focus. (D-F) One-cell embryo in metaphase. (G-I) Two-cell in which AB (left cell) is in anaphase and P1 is in metaphase. (J-L) Four-cell embryo in which the AB cells (top left cells) are in prophase and EMS and P2 are in interphase. (M-U) Co-localization of OOC-3 and HDEL staining. (M-O) One-cell embryo in anaphase. (P-R) Two-cell embryo in early interphase. (S-U) Two-cell embryo in which AB is in anaphase and P1 is in metaphase. The stage of the cell cycle was determined by DAPI staining (not shown) in all cases. Scale bar: 10 µm.

View larger version (86K): [in a new window]   Fig. 5. OOC-5 localization depends on OOC-3 in embryos, but OOC-3 localization does not require OOC-5. (A-I) Confocal micrographs of ooc-5 (it145) embryos double labeled with anti-OOC-3 (A,D,G) and anti-HDEL antibodies (B,E,H). Merged images show the extent of co-localization (C,F,I). (A-C) One-cell prophase embryo with one pronucleus visible; the other pronucleus is out of the plane of focus. (D-F) One-cell embryo in anaphase. (G-I) Two-cell embryo in interphase. (J-R) Confocal micrographs of ooc-3 (mn241) embryos double labeled with anti-OOC-5 (J,M,P) and anti-HDEL antibodies (K,N,Q). Merged images show the extent of co-localization (L,O,R). (J-L) One-cell prophase embryo with one pronucleus visible. (M-O) One-cell embryo in anaphase. (P-R) Two-cell interphase. All confocal images were obtained using the same settings as for the wild-type embryos in Fig. 4. Scale bar: 10 µm.

View larger version (131K): [in a new window]   Fig. 6. OOC-5 and OOC-3 localization in germlines and intestinal cells. Confocal micrographs of germlines and intestinal cells double labeled with either anti-OOC-5 (A,E,I,M) or anti-OOC-3 antibodies (C,G,K,O) and anti-HDEL antibodies (B,D,F,H,J,L,N,P). (A-H) Oocytes in proximal germlines of wild type (N2) and mutant worms. (I-P) Intestinal cells. Scale bar: 10 µm.

View larger version (84K): [in a new window]   Fig. 7. Distribution of GLP-1 in embryos. Digital images of wild-type (A-B) and ooc-5 embryos (C-D) labeled with anti-GLP-1 antibodies. (A) Two-cell wild-type embryo with GLP-1 localization in the AB cell. (B) Four-cell wild-type embryo with GLP-1 localization at the plasma membrane and in the cytoplasm of ABa and ABp. (C) Two-cell ooc-5 embryo with GLP-1 localization in the AB cell. (D) Four-cell ooc-5 embryo with GLP-1 localization at the plasma membrane and in the cytoplasm of ABa and ABp. Scale bar: 10 µm.

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