Kajiura Hiroko, Yamashita Masakane, Katsu Yoshinao, Nagahama Yoshitaka
Laboratory of Reproductive Biology, Department of Developmental Biology, National Institute for Basic Biology, Okazaki 444, Japan.
Laboratory of Molecular and Cellular Interactions, Division of Biological Sciences, Graduate School of Science, Hokkaido University, Sapporo 060, Japan.
Dev Growth Differ. 1993 Dec;35(6):647-654. doi: 10.1111/j.1440-169X.1993.00647.x.
We have isolated a cdc2 cDNA from a library constructed from immature goldfish oocytes. The isolated clone has a PSTAVR sequence, instead of the PSTAIR sequence common to cdc2 in other species. Its product was characterized by monoclonal antibodies against its C-terminal amino acid sequence. The antibodies recognized an anti-PSTAIR-reactive 35 kDa protein in immature oocyte extracts, which was not recognized by anti-goldfish cdk2 antibody. In addition to the 35 kDa cdc2, mature oocytes contained a 34 kDa cdc2, which was a component of MPF purified from carp eggs. Upon gel filtration column, the 35 kDa cdc2 migrated at monomeric position, while the 34 kDa cdc2 migrated at around 100 kDa, where cyclin B also comigrated. These results strongly suggest that the 35 kDa protein is monomeric inactive cdc2, while the 34 kDa protein is cyclin B-bound active cdc2. The finding that the 35 kDa inactive cdc2 does not form a complex with any other proteins in immature oocytes is in contrast to the situation in Xenopus and starfish, in which cdc2-cyclin B complex exists already as pre-MPF in immature oocytes.
我们从由未成熟金鱼卵母细胞构建的文库中分离出了一个cdc2 cDNA。分离出的克隆具有PSTAVR序列，而非其他物种中cdc2常见的PSTAIR序列。其产物通过针对其C末端氨基酸序列的单克隆抗体进行表征。这些抗体在未成熟卵母细胞提取物中识别出一种与抗PSTAIR反应的35 kDa蛋白，而抗金鱼cdk2抗体无法识别该蛋白。除了35 kDa的cdc2，成熟卵母细胞中还含有一种34 kDa的cdc2，它是从鲤鱼卵中纯化的MPF的一个组成部分。在凝胶过滤柱上，35 kDa的cdc2在单体位置迁移，而34 kDa的cdc2在约100 kDa处迁移，细胞周期蛋白B也在该位置共同迁移。这些结果有力地表明，35 kDa的蛋白是单体无活性的cdc2，而34 kDa的蛋白是与细胞周期蛋白B结合的活性cdc2。在未成熟卵母细胞中35 kDa无活性的cdc2不与任何其他蛋白形成复合物这一发现，与非洲爪蟾和海星的情况相反，在这两种生物中，cdc2 - 细胞周期蛋白B复合物在未成熟卵母细胞中已经以前体MPF的形式存在。
