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First published online 5 October 2004
doi: 10.1242/jcs.01417

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Caenorhabditis elegans UBC-2 functions with the anaphase-promoting complex but also has other activities

Tyralynn Frazier^1,*, Diane Shakes^2,*, Uma Hota¹ and Lynn Boyd^1,

¹ Department of Biological Sciences, University of Alabama in Huntsville, Huntsville, AL 35899, USA
² Department of Biology, College of William and Mary, PO Box 8795, Williamsburg, VA 23187, USA

View larger version (42K): [in a new window]   Fig. 1. RNAi of ubc-2 and apc-11 show indistinguishable embryonic phenotypes in Caenorhabditis elegans. Several embryos from each RNAi treatment are shown. The left panels show DAPI staining and the right panels show pHisH3 staining to detect metaphase figures. These embryos have all failed to complete the first cell division and the meiotic arrest phenotype is apparent in both sets of RNAi. Representative oocyte metaphase chromosomes are indicated with arrows and sperm chromatin is indicated by carets. During the meiotic divisions of the oocyte chromosomes, the quiescent, hypercondensed sperm chromatin mass does not stain with pHisH3 antibodies (Golden et al., 2000).

View larger version (48K): [in a new window]   Fig. 2. Mitotic effects in C. elegans gonads of ubc-2 and mat-1/apc-3 mutant combinations. Distal gonads were stained with pHisH3 antibody after 40 hours at 25°C. Significant increase in pHisH3 staining is seen in the mutant combinations with ubc-2(RNAi) but not apc-11(RNAi) indicating an increase in the number of M-phase chromosomes. Therefore, ubc-2 and mat-1 interact genetically to affect progression through the cell cycle in this region of the gonad.

View larger version (21K): [in a new window]   Fig. 3. In vitro C. elegans ubiquitination assays with APC-11 and UBC-2. Ubiquitination reactions were carried out as described in Materials and Methods. (A) Reactions were performed with GST-APC-11 and UBC-2. Ubiquitination reactions were probed with anti-HA antibody. Activity depends on the presence of both E1 and E2 enzymes, as well as APC-11. In the last lane, C. elegans RBX-1 is used as a positive control with the human E2, UbcH5c. (B) APC-11 that contains a mutation in a conserved residue of the RING finger shows no ubiquitination activity. The Coomassie blue-stained gel on the right shows purified wild-type and mutant GST-APC-11 fusion proteins.

View larger version (29K): [in a new window]   Fig. 4. APC-11 functions with UBC-2, but not other Ubcs in C. elegans. (A) To test the specificity of the E2-E3 interaction, in vitro ubiquitination reactions of GST-APC-11 together with nine different Ubcs were carried out and probed with anti-HA antibody. APC-11 activity was only seen with UBC-2. Some activity was also observed with UBC-1, however, the significance of this result is doubtful given that UBC-1 exhibited a similar level of activity with GST alone (data not shown). In addition, UBC-1 activity did not generate high molecular weight species characteristic of the robust ubiquitination activity seen with UBC-2. The lower Coomassie blue-stained gel shows the different purified Ubcs. Lane numbers correspond to the different Ubcs listed on the left. (B) In vitro ubiquitination activity of Ubcs with RBX-1. Lane numbers correspond to the different Ubcs listed in A. Only UBC-7 and UEV-2 do not show activity with RBX-1. UBC-20 was not included owing to low amounts of protein in those preparations. (C) Phylogenetic tree of the different Ubcs relevant to this study. The tree shows the relatedness of worm Ubcs with Ubc-X from Xenopus, E2-C from clam, UbcH10, UbcH5c and Ubc4 from humans, and Sc Ubc4p from Saccharomyces cerevisiae. The tree was generated using DNASTAR MegAlign software. Sequence data for the worm Ubcs and UEV-2 (a Ubc variant that lacks the active site cysteinyl residue) were obtained from the WormBase web site (WormBase, 2003, http://www.wormbase.org/).

View larger version (63K): [in a new window]   Fig. 5. C. elegans gonad phenotypes associated with ubc-2 RNAi. (A) The top row shows Nomarski images of gonads after 34 hours at 25°C. Gonads from ubc-2(RNAi) animals appear abnormal. The lower panels show DAPI-stained gonads. Expansion of the transition zone can be seen in ubc-2(RNAi) gonads. In both N2 and mat-1(ax161) animals, pachytene nuclei can be seen in the right hand side of the panel. Pachytene nuclei are rare or significantly displaced towards the proximal side of the gonad in ubc-2(RNAi) animals and extend nearly to the end of the normal pachytene region. Diakinetic nuclei look normal with six paired homologs. Significant enhancement of the mitotic metaphase plate phenotype can be seen in the mat-1(ax161); ubc-2(RNAi) gonads. (B) Extended RNAi with ubc-2 results in an Emo phenotype. A DAPI-stained gonad from a worm that was fed ubc-2 dsRNA from the L1 stage. An excessive amount of DNA is apparent in the large oocytes of the proximal region of the gonad (right side).

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