MITOTIC CHECKPOINT GENE MAD2 IN VERTEBRATES

生化实验表明，在后生动物中，有丝分裂检查点基因MAD2是有丝分裂检查点机制的重要组成部分。MAD2是该机制的一个组成部分，如果不是所有的染色体都附着在有丝分裂纺锤体上，它会在中期到后期过渡之前阻止细胞。这项研究的主要目的是确定哺乳动物细胞中MAD2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、基因组不稳定和/或肿瘤进展，无论是在正常生长条件下还是当检查点装置受到有丝分裂纺锤体抑制剂的挑战时。脊椎动物中有丝分裂检查点机制的任何组成部分的缺失是否会导致染色体不稳定或细胞转化，目前还没有得到证实。通过基因打靶使MAD2基因的一个拷贝失活的小鼠和人类细胞将进一步分析有丝分裂检查点途径中的缺陷。在有丝分裂过程中，当有丝分裂纺锤体被抑制时，将确定这些细胞是否能够适当地停止，如果不是，这种失败对整个染色体损失率和致瘤特性的后果。此外，通过引入显性负的MAD2等位基因或有条件地灭活第二个MAD2等位基因，MAD2在小鼠和人类细胞中的剂量都将减少。这些细胞可能有更严重的有丝分裂检查点缺陷，也将被分析染色体稳定性的变化和致癌特性的变化。通过这种方式，有丝分裂检查点通路在哺乳动物癌症启动和/或进展中的作用将被评估。

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.