Functional analysis of Cyclin A/Cdk2 for chromosome condensation

我们已经证明，非洲爪哇周期蛋白B2通过其细胞质保留信号(CRS)区域参与了双极纺锤体的形成。然而，Cyclin B2 CRS区存在的NES样序列(NEL)可能参与了双极纺锤体的形成。我们在这里表明，细胞周期蛋白B2实际上是通过其NEL从细胞核输出的，但细胞周期蛋白B2 CRS区域的过度表达，具有突变的NEL，仍然抑制卵母细胞中双极纺锤体的形成。相反，缺乏C端7个氨基酸的细胞周期蛋白B2CRS区域的过度表达不再抑制卵母细胞或胚胎中双极纺锤体的形成。这些结果有力地表明，在减数分裂和有丝分裂过程中，细胞周期蛋白B2的CRS区，特别是其C端的7个酸性残基，是形成双极纺锤体所必需的。在非洲爪哇卵母细胞中，细胞周期调节因子和前MPF被储存起来，为卵母细胞成熟带来特定的细胞周期。我们研究了这些细胞周期调节因子的表达模式以及卵子发生过程中前MPF的形成。Cdc2和Cyclin B2在I期卵母细胞中已经存在，在I期卵母细胞中也检测到前MPF的形成。在卵子发生早期就合成了MPF的负调控因子Myt1和Chk1。相反，MPF、MEK、MAPK和CDC25C等正调控因子主要在卵子发生后期合成。Northern印迹分析表明，这些细胞周期调节因子的合成受翻译控制。

We have shown that Xenopus cyclin B2 is involved in bipolar spindle formation through its cytoplasmic retention signal (CRS) region. However, an NES-like sequence (NELS) present in the CRS region of cyclin B2 might be involved in bipolar spindle formation. We show here that cyclin B2 is actually exported from the nucleus via its NELS, but that overexpression of the cyclin B2 CRS region, having a mutated NELS, still inhibits bipolar spindle formation in oocytes. In contrast, overexpression of the cyclin B2 CRS region lacking its C-terminal seven amino acids no longer inhibits bipolar spindle formation in oocytes or embryos. These results suggest strongly that the CRS region, especially its C-terminal seven acidic residues, of cyclin B2 is required for bipolar spindle formation in both the meiotic and mitotic cell divisions.In full-grown Xenopus oocytes, cell-cycle regulators and pre-MPF are stored ready to bring about a specific cell cycle for oocyte maturation. We examined the expression pattern of these cell-cycle regulators as well as pre-MPF formation during oogenesis. Cdc2 and Cyclin B2 were already present in stage I oocytes and pre-MPF formation was also detected in stage I oocytes. Some negative regulators of MPF, Myt1 and Chk1, were synthesized early in oogenesis. In contrast, positive regulators of MPF, MEK, MAPK and Cdc25C, were mainly synthesized late in oogenesis. Northern blotting analysis suggested that the synthesis of these cell-cycle regulators was controlled by translation.