Cell cycle dependent expression of Plk1 in synchronized porcine fetal fibroblasts
Cell cycle dependent expression of Plk1 in synchronized porcine fetal fibroblasts
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DOI：
10.1002/mrd.10289
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发表时间：
2003-07-01
影响因子：
2.5
通讯作者：
Niemann, H
中科院分区：
文献类型：
--
作者：
Anger, M;Kues, WA;Niemann, H
polo样激酶（Plk）家族的酶在控制有丝分裂的途径中很活跃。我们以胎儿成纤维细胞为来源克隆了Plk1、Plk2和Plk3的猪同源基因cDNA片段。这三个部分cdna与小鼠和人类的对应序列具有高度的同源性，并且包含Polo盒子，这是所有Polo激酶的特征结构域。采用RT-PCR和核糖核酸酶保护实验分析同步猪胎儿成纤维细胞不同细胞周期点Plk1 mRNA的表达水平。Plk1 mRNA在G0和G1期几乎检测不到，在S期增加，在G2/M期过渡后达到峰值。制备了一种针对体外表达的猪Plk1蛋白片段的单克隆抗体，用于检测Plk1蛋白水平的表达变化。免疫印迹法在S期开始时首次检测到PIk1蛋白，在G2/M过渡后最高。总之，Plk1在猪中的表达模式与报道的其他物种相似。PIk1 mRNA和蛋白的缺失似乎是G0/G1的一个很好的标记，因此可以选择基于核转移的体细胞克隆供体细胞。(C) 2003 Wiley-Liss, Inc。
Enzymes of the Polo-like kinase (Plk) family are active in the pathways controlling mitosis in several species. We have cloned cDNA fragments of the porcine homologues of Plk1, Plk2, and Plk3 employing fetal fibroblasts as source. All three partial cDNAs showed high sequence homology with their mouse and human counterparts and contained the Polo box, a domain characteristic for all Polo kinases. The expression levels of Plk1 mRNA at various points of the cell cycle in synchronized porcine fetal fibroblasts were analyzed by both RT-PCR and the ribonuclease protection assay. Plk1 mRNA was barely detectable in G0 and G1, increased during S phase and peaked after the G2/M transition, A monoclonal antibody was generated against an in vitro expressed porcine Plk1-protein fragment and used to detect changes in Plk1 expression at the protein level. PIk1 protein was first detected by immunoblotting at the beginning of S phase and was highest after the G2/M transition. In summary, the Plk1 expression pattern in the pig is similar to that reported for other species. The absence of PIk1 mRNA and protein appears to be a good marker for G0/G1 and thus for the selection of donor cells for nuclear transfer based somatic cloning. (C) 2003 Wiley-Liss, Inc.