In the early embryonic cell cycle, exit from M phase is immediately followed by entry into S phase without an intervening gap phase. To understand the regulatory mechanisms for the cell cycle ...transition from M to S phase, we examined dependence on Cdc2 inactivation of cell-cycle events occurring during the M-S transition period, using Xenopus egg extracts in which the extent of Cdc2 inactivation at M phase exit was quantitatively controlled. The result demonstrated that MCM binding to and the initiation of DNA replication of nuclear chromatin occurred depending on the decrease of Cdc2 activity to critical levels. Similarly, we found that Cdc2 inhibitory phosphorylation and cyclin B degradation were turned on and off, respectively, depending on the decrease in Cdc2 activity. However, their sensitivity to Cdc2 activity was different, with the turning-on of Cdc2 inhibitory phosphorylation occurring at higher Cdc2 activity levels than the turning-off of cyclin B degradation. This means that, when cyclin B degradation ceases at M phase exit, Cdc2 inhibitory phosphorylation is necessarily activated. In the presence of constitutive synthesis of cyclin B, this condition favors the occurrence of the Cdc2 inactivation period after M phase exit, thereby ensuring progression through S phase. Thus, M phase exit and S phase entry are coordinately regulated by the Cdc2 activity level in the early embryonic cell cycle.
We have studied the cytoplasmic mechanism that induces metaphase chromosome condensation in cell‐free Xenopus egg extracts. To analyze the mechanism responsible for inducing chromosome condensation ...separately from those responsible for sperm chromatin remodeling and nuclear envelope disassembly, we used Xenopus sperm chromatin that had already been remodeled to nucleosomal chromatin by incubating demembranated sperm with egg extracts added with lysolecithin. We found that inhibition of cyclin B‐Cdc2 with butyrolactone I abolished chromosome condensation of the remodeled sperm chromatin by M‐phase egg extracts, but incubation of the chromatin with active cyclin B‐Cdc2 alone did not induce chromosome condensation, indicating a requirement for cytoplasmic factor(s) in addition to cyclin B‐Cdc2 for the induction of chromosome condensation. We further demonstrated that if the cyclin B‐Cdc2‐dependent phosphorylation state was protected against dephosphorylation by a preincubation of M‐phase extracts with ATP‐γ‐S, chromosome condensation and phosphorylation of chromosomal histone H1 occurred even when extracts were depleted of cyclin B‐Cdc2 activity. The chromosome condensation seen in the absence of cyclin B‐Cdc2 was completely inhibited with another protein kinase inhibitor, 6‐dimethylaminopurine, implying that a protein kinase other than cyclin B‐Cdc2 was involved in the induction of chromosome condensation. These results strongly suggest that a cyclin B‐Cdc2‐dependent protein kinase cascade is involved in inducing chromosome condensation and the phosphorylation of chromosomal histone H1.
We have studied the cytoplasmic mechanism that induces metaphase chromosome condensation in cell-free
Xenopus egg extracts. To analyze the mechanism responsible for inducing chromosome condensation ...separately from those responsible for sperm chromatin remodeling and nuclear envelope disassembly, we used
Xenopus sperm chromatin that had already been remodeled to nucleosomal chromatin by incubating demembranated sperm with egg extracts added with lysolecithin. We found that inhibition of cyclin B-Cdc2 with butyrolactone I abolished chromosome condensation of the remodeled sperm chromatin by M-phase egg extracts, but incubation of the chromatin with active cyclin B-Cdc2 alone did not induce chromosome condensation, indicating a requirement for cytoplasmic factor(s) in addition to cyclin B-Cdc2 for the induction of chromosome condensation. We further demonstrated that if the cyclin B-Cdc2-dependent phosphorylation state was protected against dephosphorylation by a preincubation of M-phase extracts with ATP-γ-S, chromosome condensation and phosphorylation of chromosomal histone H1 occurred even when extracts were depleted of cyclin B-Cdc2 activity. The chromosome condensation seen in the absence of cyclin B-Cdc2 was completely inhibited with another protein kinase inhibitor, 6-dimethylaminopurine, implying that a protein kinase other than cyclin B-Cdc2 was involved in the induction of chromosome condensation. These results strongly suggest that a cyclin B-Cdc2-dependent protein kinase cascade is involved in inducing chromosome condensation and the phosphorylation of chromosomal histone H1.
We have investigated the molecular target of an antitumor agent ICRF-193, a bisdioxopiperazine derivative, inin vitrochromosome condensation system ofXenopusegg extract (XEE), where DNA topoisomerase ...II was previously demonstrated to play a crucial role. DemembranatedXenopussperm head chromatin is converted to metaphase chromosome-like structure in XEE in two steps,i.e.,swelling of the chromatin followed by condensation of chromosome. When ICRF-193 was added to the reaction, swelling of the chromatin was not affected but chromosome condensation was completely blocked. This blockade was reversed by exogenous supplement of calf thymus topoisomerase II, which was in turn neutralized by anti-topoisomerase II monoclonal antibody. These results demonstrate that topoisomerase II is the molecular target of the drug ICRF-193.
We have investigated the molecular target of an antitumor agent ICRF-193, a bisdioxopiperazine derivative, in in vitro chromosome condensation system of Xenopus egg extract (XEE), where DNA ...topoisomerase II was previously demonstrated to play a crucial role. Demembranated Xenopus sperm head chromatin is converted to metaphase chromosome-like structure in XEE in two steps, i.e., swelling of the chromatin followed by condensation of chromosome. When ICRF-193 was added to the reaction, swelling of the chromatin was not affected but chromosome condensation was completely blocked. This blockade was reversed by exogenous supplement of calf thymus topoisomerase II, which was in turn neutralized by anti-topoisomerase II monoclonal antibody. These results demonstrate that topoisomerase II is the molecular target of the drug ICRF-193.
In order to obtain the cytological basis for the periodic flattening and rounding‐up of activated amphibian eggs, the surface ultrastructure and the cortical microfilament organization were studied ...in
Xenopus laevis
. Scanning electron microscopy (SEM) of the egg surface revealed that the density of microvilli at the animal pole region decreased significantly when the periodic flattening started, but increased again concomitantly with the commencement of the rounding‐up. Isolated pieces of the cortices stained with rhodamine‐phalloidin exhibited the periodic disorganization and reorganization of a meshwork with bright dots probably corresponding to microvilli, in good synchrony with the decrease and increase of the microvilli density. Study of appropriate batches of eggs in which the moving front of surface contraction waves (SCWs; 1) can be localized revealed that the decrease and increase of the microvilli density correspond to SCW‐1 and ‐2, respectively. SEM and the cytochemical examination of the eggs from which the germinal vesicle (GV) had been removed revealed that none of these changes occurred in the enucleated eggs. These observations suggest that the GV‐dependent regulation of the microfilament organization in an egg cortex constitutes the cytological basis for the SCWs and for the periodic flattening and rounding‐up of denuded eggs.
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