MITOTIC CHECKPOINT GENE MAD2 IN VERTEBRATES

有丝分裂检查点基因Mad 2已通过生化实验显示在后生动物中是有丝分裂检查点装置的重要部分。Mad 2是一种机制的组成部分，如果所有的染色体都没有附着在有丝分裂纺锤体上，则Mad 2会在中期向后期过渡之前阻止细胞。这项研究的主要目的是确定在正常生长条件下或当检查点装置受到有丝分裂纺锤体抑制剂的挑战时，哺乳动物细胞中Mad 2功能的完全或部分丧失是否会导致有丝分裂检查点控制的丧失、基因组不稳定性和/或肿瘤进展。目前尚未证实脊椎动物中有丝分裂检查点机制的任何组分的丢失是否会导致染色体不稳定或细胞转化。将进一步分析通过基因靶向失活的Mad 2基因的一个拷贝的小鼠和人细胞在有丝分裂检查点途径中的缺陷。将确定当有丝分裂纺锤体被抑制时，这些细胞是否可以在有丝分裂期间适当地停滞，如果不能，则确定这种失败对整个染色体丢失率和致瘤性的后果。此外，通过引入显性负性Mad 2等位基因或第二个Mad 2等位基因的条件性失活，可降低小鼠和人细胞中Mad 2的剂量。这些细胞可能具有更严重的有丝分裂检查点缺陷，也将分析染色体稳定性的变化和致瘤性的改变。以这种方式，将评估有丝分裂检查点途径在哺乳动物中癌症起始和/或进展中的作用。

The mitotic checkpoint gene Mad2 has been shown in metazoans to be an important part of the mitotic checkpoint apparatus through biochemical experiments. Mad2 is a component of the machinery which arrests a cell prior to the metaphase to anaphase transition if all the chromosomes are not attached to the mitotic spindle apparatus. The main goal of the research being proposed is to determine if complete or partial loss of Mad2 function in mammalian cells leads to a loss of mitotic checkpoint control, genomic instability and/or tumor progression either under normal growth conditions or when the checkpoint apparatus is challenged with mitotic spindle inhibitors. It has not yet been demonstrated whether the loss of any component of the mitotic checkpoint mechanism in vertebrates leads to chromosome instability or cellular transformation. Mice and human cells that have one copy of the Mad2 gene inactivated by gene targeting will be further analyzed with respect to defects in the mitotic checkpoint pathway. It will be determined whether such cells can arrest appropriately during mitosis when the mitotic spindle is inhibited, and if not, the consequences of this failure on whole chromosome loss rates and tumorigenic properties. In addition, the dosage of Mad2 will be decreased in both mouse and human cells by the introduction of dominant negative Mad2 alleles, or conditional inactivation of the second Mad2 allele. These cells, likely to have a more severe mitotic checkpoint defect, will also be analyzed for changes in chromosome stability and alterations in tumorigenic properties. In this way, the role of the mitotic checkpoint pathway in cancer initiation and/or progression in mammals will be assessed.