Cloning of a novel tumor suppressor gene in Wilms' tumor associated with chromosome instability

镶嵌杂色非整倍体1A型(MVA1A型)是一种罕见的常染色体隐性遗传不稳定综合征，与肾母细胞瘤相关。我们设计这项研究是为了寻找该综合征的致病基因。为此，我们首先采用了互补克隆策略。将人乳腺逆转录病毒文库分为60个小池文库，每个小池文库包含3000个独立克隆。所有小池文库均被导入永生化的MVA1A细胞。用后期抑制剂莫那曲醇处理细胞24小时，每3天10次。我们获得了16个候选基因。第一次筛选，但每一个都没有显示出与MVA1A表型的明显互补。我们还对MVA1A细胞的有丝分裂进程进行了详细的研究。我们发现了接下来的两个新发现：(1)当MVA1A细胞暴露在纺锤体.d聚合剂中时，细胞被纺锤体检查点暂时阻止在有丝分裂中，然后覆盖这个检查点。(2)姐妹着丝粒在分裂后期前解离。这些结果表明，MVA1a的主要缺陷存在于着丝粒着丝粒-着丝点功能。然后，我们对MVA1ACENP-A、CENP-C、HP-1β、NDC80、NuF2、AuroraB、Survivin、INCENP等几个已知的着丝粒着丝粒动核基因进行了突变分析。到目前为止，还没有检测到负责任的突变。

Mosaic variegated aneuploidy type 1A (MVA1A) is a rare autosomal recessive chromosome instability syndrome associated with Wilms'tumor. We designed this study to identify the responsible gene of this syndrome.For this purpose, first, we used complementation cloning strategy. The retroviral library derived human mammary gland was divided into 60 small pool libraries that each including 3000 independent clones. All of the small pool libraries were transfected to immortalized MVA1A cells. Transfected cells were exposed with monastrol, one of anaphase inhibitor, for 24 hours ten times every three days. We obtained 16 candidate genes in. the first screening, but each of them didn't show no clear complementation against the MVA1A phenotype.We also underwent detailed studies about the progression of mitosis in MVA1A cells. We found next two new findings ; (1) When MVA1A cells were exposed with spindle.d epolymerizing agents ; the cells transiently arrested in mitosis by spindle checkpoint, and then overrided this checkpoint. (2) The sister centromeres were dissociated before anaphase. These results suggested that the primary defects of MVA1A were existed in centromere-kinetocore function. Then, we underwent mutation analysis of several known centromere-kinetocore genes, such as CENP-A, CENP-C, HP-1β, Ndc8O, Nuf2, AuroraB, Survivin, INCENP, in MVA1A. No responsible mutation have detected until now.