Defective rRNA processing and p53 activation in <i>hkc</i> mutants.

<p>(A–E) Detection of rRNA processing intermediates by Northern blot hybridization. Total cellular RNAs (0.5 µg per lane for probes A, C, and E; 0.1 µg per lane for probes B and D) in 7-dpf embryos from WT, siblings (Sib), and <i>hkc</i> mutants were electrophoresed in agarose gels and was hybridized with indicated probes (panels A, B, and D). Images of methylene blue (MB) staining are also shown. Indicated amount of total cellular RNAs in 7-dpf embryos from WT, siblings (Sib), and <i>hkc</i> mutants were electrophoresed in polyacrylamide gel and hybridized with probe C (panel E). Predicted structures of intermediates a-c and positions of probes A–E are drawn (panel C). In panel E, the <i>hkc</i> lane shows an increase in slowly electrophoresed signal (designated as X), perhaps corresponding to the mixture of the intermediates described in panel C. The amount of housekeeping <i>cytoplasmic actin</i> mRNA measured by quantitative RT-PCR was not significantly different among same-weight total cellular RNAs isolated from WT, Sib, and <i>hkc</i> mutants. Along with data showing that the intensities of the bands for 28S, 18S, and 5.8S rRNAs were comparable among these three groups of cells, these results suggest that the total amounts of rRNAs were comparable among these three groups of the cells. (F) Quantitative RT-PCR analysis of indicated genes in whole bodies of 7-dpf WT (open bars) and <i>hkc</i> mutants (closed bars). Results represent averages and standard errors of four independent measurements. Asterisks, p<0.05. (G) Phenotypes of <i>WDR55^hkc/hkcp53^Y186X/Y186X</i> embryos. Whole-mount <i>in situ</i> hybridization of <i>rag1</i> was carried out with 5- to 7-dpf embryos obtained from <i>WDR55^+/hkcp53^Y186X/Y186X</i>×<i> WDR55^+/hkcp53^Y186X/Y186X</i> crosses or <i>WDR55^+/hkcp53^+/Y186X</i>× <i>WDR55^+/hkcp53^+/Y186X</i> crosses. (H) Quantitative RT-PCR analysis of indicated genes in whole bodies of 7-dpf WT (open bars) and <i>p53^Y186X/Y186X</i> mutants (closed bars). Results represent averages and standard errors of four independent measurements. Asterisks, p<0.05.