Characterization of the genes encoding for MAD2 homologues in wheat
Characterization of the genes encoding for MAD2 homologues in wheat
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DOI：
10.1023/b:chro.0000045760.42880.8c
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发表时间：
2004-01-01
影响因子：
2.6
通讯作者：
Nasuda, S
中科院分区：
文献类型：
--
作者：
Kimbara, J;Endo, TR;Nasuda, S
MAD 2（mitotic arrest deficiency 2）是一个高度保守的蛋白质，参与纺锤体检查点控制。MAD 2在分裂前期定位于纺锤体不附着的动粒上，在分裂中期从纺锤体附着的动粒上溶解。本研究从六倍体小麦中分离了MAD 2的同源基因。第2组染色体长臂上的3个同源异型基因在编码区的核苷酸序列相似性约为99%。这三个同源基因的内含子-外显子结构也是保守的，与拟南芥MAD 2基因具有高度的相似性。所有三个同源物在根和穗中转录，但在叶中不转录。我们产生了针对具有来自小麦MAD 2同源物的cDNA序列的氨基酸序列的多肽的抗体。使用这些抗体，我们发现小麦根尖细胞中的MAD 2在细胞周期中的位置和数量发生变化，与人类MAD 2的报告相似。当根尖用秋水仙素（一种微管不稳定药物）处理时，在所有中期染色体的着丝粒处观察到强烈的免疫染色信号，但在未处理的染色体中没有观察到信号。因此，小麦MAD 2蛋白可以作为小麦中期染色体功能着丝粒的一个很好的标记。
MAD2 (mitotic arrest deficient 2) is a highly conserved protein involving in spindle checkpoint control. MAD2 locates at spindle-unattached kinetochores during prophase and dissolves from spindle-attached kinetochores towards metaphase. In this study, we isolated homologous genes encoding for MAD2 from hexaploid wheat. The three homoeologous genes on the long arms of the group-2 chromosomes shared approximately 99% similarity of the nucleotide sequence in coding regions. The intron-exon structures of the three homoeologues were also conserved, showing high similarities to that of the Arabidopsis MAD2 gene. All three homoeologues were transcribed in roots and spikes but not in leaves. We generated antibodies against the polypeptides with amino acid sequences derived from the cDNA sequences of the wheat MAD2 homologues. Using these antibodies, we found MAD2 in wheat root-tip cells to change in location and amount through the cell cycle, similar to those reported for human MAD2. Intense immunostaining signals were observed at the centromeres of all metaphase chromosomes when root-tips were treated with colchicine, a microtubule-destabilizing drug, but no signals were observed in untreated chromosomes. Thus, the wheat MAD2 protein could be a good marker for the functional kinetochores of metaphase chromosomes in wheat.