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First published online January 25, 2006
doi: 10.1242/10.1242/dev.02235

Development 133, 621-629 (2006)
Published by The Company of Biologists 2006
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URI-1 is required for DNA stability in C. elegans

Christine T. Parusel1, Ekaterini A. Kritikou2,*, Michael O. Hengartner2, Wilhelm Krek1,{dagger} and Monica Gotta3,{dagger}

1 Eidgenoessische Technische Hochschule Zuerich, Institute of Cell Biology, CH-8093 Zuerich, Switzerland.
2 University of Zuerich, Institute of Molecular Biology, CH-8057 Zuerich, Switzerland.
3 Eidgenoessische Technische Hochschule Zuerich, Institute of Biochemistry, CH-8093 Zuerich, Switzerland.


Figure 1
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  		Fig. 1. Characterization of uri-1 in C. elegans. (A) Genomic structure of C. elegans uri-1 (C55B7.5) and uri-1 deletion mutant (tm939). URI-1 encodes a 446 amino acid protein with a putative prefoldin domain (PFD), a direct binding site for the common subunit of all three RNA polymerases RPB5 (RPB5) and the URI-1 box depicted below. (B) Images from wild-type adult hermaphrodites, uri-1(RNAi)F1 and homozygous uri-1(tm939) adult hermaphrodites raised at 25°C and stained with DAPI. Arrows indicate haploid sperm and arrowheads indicate spermatids. Anterior is towards the left and dorsal is towards the top throughout. Scale bar 10 µm. (C) Northern blot was performed as described in the Materials and methods and shows that uri-1 is a germline-enriched transcript expressed in both types of gametes. Ribosomal RNA was used as loading control.

 

Figure 2
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  		Fig. 2. uri-1(lf) mutants have defects in germ cell proliferation. (A) Number of germ nuclei per gonad arm in uri-1(RNAi)F1 (n=25) and wild-type (n=25) worms. Progeny of staged animals were synchronized by letting the P0 generation to lay eggs for 2 hours, fixed at the indicated time points and stained with DAPI. Germ cells identified by their DNA morphology were counted. Error bars represent s.d. (B) Images of gld-2(If) gld-1(If) (top) and gld-2(If) gld-1(If) uri-1(RNAi)F1 (bottom) gonads. Loss of URI-1 results in reduction of mitotic germ cells. For both images, anterior is towards the left and dorsal towards the top. Scale bar: 10 µm.

 

Figure 3
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  		Fig. 3. Loss of URI-1 blocks cell cycle progression in the C. elegans germline. (A) (Left) Quantification of G2/M phase cells in untreated wild-type (n=35, dark) and uri-1(RNAi)F1 (n=35, grey) synchronized adult hermaphrodites (*P=5.2x10-5). Error bars represent s.d. (Right) DAPI and PH3 staining of the distal part of the germline of a uri-1(RNAi)F1 and a wild-type worm. The arrowheads indicate mitotic germline cells with apparently condensed chromosomes and different extent of enlargement. This DNA morphology accumulates in germlines upon loss of uri-1 function and resembles prometaphases which are not recognized by the PH3 antibody (Cy3). Scale bar: 10 µm. (B) (Right) Staining with anti-phospho-histone H3 antibody (Cy3) of germ cell nuclei (visualized with DAPI) in the distal region of the gonad of the indicated strains. (Left) PH3-positive nuclei were counted per gonad arm in synchronized adults uri-1(tm939) homozygous (n=24), uri-1(RNAi)F1 (n=65) and wild type (n=65, Student's test *P=7.9x10-8, #P=1.0x10-4). Scale bar 10 µm. Error bars indicate s. d. (C) Anti-PH3 (Cy3) and DAPI staining of a homozygous uri-1(lf) mutant and wild-type gonads. Arrowheads indicate sperm. Scale bar: 10 µm. Anterior is towards the left and dorsal is towards the top.

 

Figure 4
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  		Fig. 4. Accumulation of DNA breaks in the adult hermaphrodite germline due to loss of uri-1 function. (A) (Top) Histograms depicting quantification of TUNEL foci in uri-1(RNAi)F1 (n=10) and wild-type (n=10) gonads at 25°C. The nuclei within a 15 nuclei diameter radius from the distal tip cell were scored. Each column represents the percentage of nuclei that contain a defined number of foci. (Bottom) TUNEL staining in wild-type and uri-1 germlines is shown. Anterior is towards the left and dorsal is towards the top. (B) Quantification (left) of the HUS-1::GFP foci in uri-1(RNAi)F1 (gray) and control RNAi (black) in hus-1(op241); unc-119(ed3); opIs34 mitotic germ cells (n=55 for each, *P=3.4x10-43) and image of germ nuclei (right) showing the relocalization of HUS-1::GFP. OpIS34 is a HUS-1::GFP integrated line. Error bars represent s.d. Scale bar: 5 µm.

 

Figure 5
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  		Fig. 5. Depletion of hus-1 and atl-1 rescues the cell cycle arrest observed in uri-1(lf) worms. Quantification of cells at the G2/M border per gonad arm in the distal region of synchronized uri-1(RNAi)F1 in the indicated genotypes in adult hermaphrodites (n=10 each). The number of germ nuclei per gonad arm was 91.3±24.0 in hus-1; uri-1(RNAi)F1 (A) and 98.8±22.9 in ced-3(lf); atl-1(RNAi); uri-1(RNAi)F1 (B). Error bars indicate s.d.

 

Figure 6
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  		Fig. 6. Effect of uri-1 depletion on meiotic cells. (A) Loss of uri-1 sensitizes meiotic germ cell to apoptosis. Apoptotic germ cell corpse numbers in the heterozygous uri-1 mutant and uri-1(RNAi)P0 animals (n=40 each) reveal a gla phenotype, which is completely suppressed by mutations in the apoptotic pathway components ced-3(lf)and ced-9(gf) (*P=3.0x10-20, #P=2.3x10-19). Error bars represent s.d. (B) Apoptotic germ cell corpse counts in wild-type and cep-1 germlines of uri-1(RNAi)P0 and control animals (n=40 each) reveal that the gla phenotype that is associated with loss of URI-1 is completely suppressed by loss of the DNA damage sensor p53 (C. elegans cep-1). Error bars represent s.d.

 

Figure 7
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  		Fig. 7. A proposed model of URI-1 function. Genetic and cell biological analysis show that URI-1 prevents DNA damage and the subsequent activation of the DNA damage signaling pathway by functioning directly or indirectly in DNA damaging processes and/or DNA repair processes. DNA damage caused by deficiency of URI-1 affects germ cells by leading to proliferation arrest in the mitotic region and p53-dependent apoptosis, as well as cell cycle arrest in the meiotic part of the germline. This phenotypes are consistent with reported read outs of the DNA-damage response (Deng et al., 2004Go; Stergiou and Hengartner, 2004Go).

 




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