Gardner R D, Poddar A, Yellman C, Tavormina P A, Monteagudo M C, Burke D J
Department of Biochemistry and Molecular Genetics, University of Virginia Medical Center, University of Virginia, Charlottesville, Virginia 22908-0733, USA.
Genetics. 2001 Apr;157(4):1493-502. doi: 10.1093/genetics/157.4.1493.
We have measured the activity of the spindle checkpoint in null mutants lacking kinetochore activity in the yeast Saccharomyces cerevisiae. We constructed deletion mutants for nonessential genes by one-step gene replacements. We constructed heterozygous deletions of one copy of essential genes in diploid cells and purified spores containing the deletion allele. In addition, we made gene fusions for three essential genes to target the encoded proteins for proteolysis (degron alleles). We determined that Ndc10p, Ctf13p, and Cep3p are required for checkpoint activity. In contrast, cells lacking Cbf1p, Ctf19p, Mcm21p, Slk19p, Cse4p, Mif2p, Mck1p, and Kar3p are checkpoint proficient. We conclude that the kinetochore plays a critical role in checkpoint signaling in S. cerevisiae. Spindle checkpoint activity maps to a discreet domain within the kinetochore and depends on the CBF3 protein complex.
我们已经在酿酒酵母中测量了缺乏动粒活性的无效突变体中纺锤体检查点的活性。我们通过一步基因替换构建了非必需基因的缺失突变体。我们在二倍体细胞中构建了必需基因一个拷贝的杂合缺失，并纯化了含有缺失等位基因的孢子。此外，我们对三个必需基因进行了基因融合，以靶向编码的蛋白质进行蛋白水解（降解等位基因）。我们确定Ndc10p、Ctf13p和Cep3p是检查点活性所必需的。相比之下，缺乏Cbf1p、Ctf19p、Mcm21p、Slk19p、Cse4p、Mif2p、Mck1p和Kar3p的细胞检查点功能正常。我们得出结论，动粒在酿酒酵母的检查点信号传导中起关键作用。纺锤体检查点活性定位于动粒内一个离散结构域，并依赖于CBF3蛋白复合体。
