Bandyopadhyay J, Bandyopadhyay A, Choi H S, Kwon H B, Kang H M
Hormone Research Center, Chonnam National University, Kwangju, 500-757, Republic of Korea.
Gen Comp Endocrinol. 2000 Feb;117(2):313-22. doi: 10.1006/gcen.1999.7420.
In order to understand the mechanism of oocyte maturation in seasonal-breeding wild frogs, we have cloned and sequenced a cDNA encoding Cdc2 kinase, a component of the maturation-promoting factor (MPF) in Rana dybowskii. About 1.2-kb cDNA was isolated by reverse transcription coupled to polymerase chain reaction (RT-PCR) and cDNA library screening. The cloned Rana Cdc2 cDNA encodes a complete open-reading frame with 302 amino acid residues, which deduce a 34-kDa protein. Homology of more than 80% was found between the deduced amino acid sequence of Rana Cdc2 and that of five phylogenetically distant organisms, and 94% identity was found between Rana and Xenopus. More importantly, the Thr14, Tyr15, and Thr161 residues, the phosphorylation sites for the activation of the enzyme, are highly conserved. In vitro-translated Rana Cdc2 cross-reacted with Xenopus p34(cdc2) antibody as shown by Western blot. Northern blot analysis showed that a 1.7-kb transcript was highly expressed in the gonads compared to other tissues, indicating the important role of Cdc2 kinase in gonads as a component of MPF. The cloned Rana Cdc2 cDNA also exhibited histone H1 kinase activity when expressed in CV-1 cells. In the present study, therefore, we have characterized the Rana Cdc2 kinase in amphibian, which will be helpful in understanding the process of oocyte maturation related to the reproduction cycle of wild frogs.
为了了解季节性繁殖野生蛙卵母细胞成熟的机制，我们克隆并测序了编码Cdc2激酶的cDNA，Cdc2激酶是东北林蛙成熟促进因子（MPF）的一个组成部分。通过逆转录聚合酶链反应（RT-PCR）和cDNA文库筛选分离出约1.2 kb的cDNA。克隆的东北林蛙Cdc2 cDNA编码一个具有302个氨基酸残基的完整开放阅读框，推导的蛋白质分子量为34 kDa。在东北林蛙Cdc2推导的氨基酸序列与5种系统发育关系较远的生物的序列之间发现了80%以上的同源性，东北林蛙与非洲爪蟾之间的同一性为94%。更重要的是，该酶激活的磷酸化位点苏氨酸14、酪氨酸15和苏氨酸161残基高度保守。蛋白质免疫印迹显示，体外翻译的东北林蛙Cdc2与非洲爪蟾p34(cdc2)抗体发生交叉反应。Northern印迹分析表明，与其他组织相比，1.7 kb的转录本在性腺中高表达，表明Cdc2激酶作为MPF的一个组成部分在性腺中具有重要作用。当在CV-1细胞中表达时，克隆的东北林蛙Cdc2 cDNA也表现出组蛋白H1激酶活性。因此，在本研究中，我们对两栖动物中的东北林蛙Cdc2激酶进行了表征，这将有助于理解与野生蛙繁殖周期相关的卵母细胞成熟过程。
