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Sea urchin embryos contain a large cytoplasmic pool of microtubular protein, from which they can regenerate cilia several times without the need for synthesizing additional tubulin. The present studies were initiated to determine if a requirement for new tubulin synthesis can be induced by depleting this pool through repeated cycles of deciliation. By the third round of deciliation, there is a 2- to S-fold increase in the rate of incorporation of r%]methionine into a- and &tubulin. To determine if this enhanced synthesis of tubulin is due to an increase in cytoplasmic levels of translatable tubulin mRNA, poly(A)-containing RNA was isolated from control and 5 x -deeiliated embryos by phenol/chloroform extraction and oligo(dT)-affinity chromatography. Translation of these RNAs in a wheat germ proteinsynthesizing system revealed a 2- to a-fold higher rate of e- andg-tubulin formation in the presence of poly(A)containing RNA from deciliated embryos. When the deciliation experiments were carried out in the presence of 100 pg/ml of actinomycin D (sufficient to inhibit RNA synthesis by greater than 95%), the enhanced rate of tubulin formation no longer occurred, although tubulin continued to be synthesized at control rates. Actinomycin D was also found to significantly inhibit the regeneration of cilia relative to nontreated organisms, but only after embryos were subjected to two to three rounds of deciliation. Since these results suggested the possible existence of control at the level of gene transcription, double label experiments were carried out to determine if the specific activity of labeled tubulin mRNA is enhanced in deciliated embryos. Deciliated and control embryos were labeled with [“HI- and [14C]uridine, respectively, and the polysomal poly(A)-containing RNA isolated and fractionated both by sodium dodecyl sulfate-sucrose gradient centrifugation and polyacrylamide gel electrophoresis. In both cases, a peak in the ‘H/14C ratio was observed with a sedimentation coefficient slightly larger than 18 S. When these RNA fractions were translated in the wheat germ cell-free system, this peak in ‘H/14C ratio was found in both cases to correspond to the peak in tub.ulin mRNA activity, suggesting that the specific activity of tubulin mRNA is elevated in deciliated embryos. These results, considered along with the actinomycin data, are consistent with the hypothesis that the increase in the level of translatable tubulin mRNA which occurs as |
