Fig. 7. MPF activation through 90 cyclin B addition to Ca²⁺, or ioniphore-activated extracts does not influence EDEN-dependent deadenylation. A. Nondegradable sea urchin 90 cyclin B was added (+) or not (-) to CSF extract at final concentration of 0.2 ng/µl and incubated for 15 minutes at 21°C to allow its association with cdk1 and the formation of a pool of stable MPF. Both extracts were then supplemented with CaCl₂ (to final concentration 0.8 mM) and incubated at 21°C for 1, 2 or 3 hours with radiolabelled transcripts Eg2-410 and Eg2-410a. RNA. Upper panel: autoradiography of the extracted RNAs separated on a urea/acrylamide 4% gel. The lane (T) corresponds to the fully adenylated radiolabelled transcripts without previous incubation with extract and precipitation. The two extracts deadenylate chimeric RNAs with similar dynamics independently from the presence or absence of 90 cyclin B. Lower panel: sperm nuclei were incubated with extracts and stained with Hoechst dye following observation in fluorescence and phase contrast. Condensed sperm chromosomes in the control CSF extract (left) and in the calcium activated CSF extract with 90 cyclin B (middle) were found at the end of the experiment, whereas decondensed nuclei were present in calcium activated CSF extract (right). This confirms that 90 cyclin B-supplemented extract was in M-phase similarly to the control, untreated CSF extract. B. Nondegradable sea urchin 90 cyclin B (final concentration 0.2 ng/µl), OA (final concentration 1 µM), or both, were added to ionophore-activated eggs extract prepared 45 minutes after egg activation. The extracts were incubated for 15 minutes at 21°C to allow association of exogenous cyclin with cdk1 and formation of stable MPF or to inactivate protein phosphatases in the case of OA. Then they were supplemented with radiolabelled Eg2-410 transcript and further incubated at 21°C for 3 hours. RNA and proteins were analysed as indicated in Materials and Methods. Upper panel: autoradiography of the extracted RNAs separated on urea/acrylamide 4% gel. The lane (T) corresponds to the fully adenylated radiolabelled transcripts without previous incubation with extract and precipitation. Untreated, as well as 90 cyclin B-supplemented extracts dedenylate Eg2-410 transcript rapidly (experiments 1 and 2), whereas OA- and OA+90 cyclin B-supplemented extracts deadenylate only slightly Eg2-410 transcript (experiments 3 and 4). Lower panel: western blot with an anti-Cdc25 antibody. The fast migrating, lowest band represents dephosphorylated (inactive) form of Cdc25. The upshifted forms of Cdc25 produced by different treatments represent phosphorylated (active) forms of the Cdc25 phosphatase. Untreated extract contains inactive Cdc25 (lane 1), whereas 90 cyclin B-supplemented extract contains activated form of the phosphatase (lane 2) indicating induction of the M-phase. OA-treated and OA+90 cyclin B-treated extracts contain phosphorylated forms of Cdc25 (lanes 3 and 4, respectively). In OA+90 cyclin B containing extract (lane 4) Cdc25 is phosphoryletd to higher degree than in OA-treated extract (lane 3), indicating a synergistic effect of 90 cyclin B and OA on Cdc25 phosphorylation.