Roberts B T, Farr K A, Hoyt M A
Department of Biology, Johns Hopkins University, Baltimore, Maryland 21218-2685.
Mol Cell Biol. 1994 Dec;14(12):8282-91. doi: 10.1128/mcb.14.12.8282-8291.1994.
Normal cell multiplication requires that the events of mitosis occur in a carefully ordered fashion. Cells employ checkpoints to prevent cycle progression until some prerequisite step has been completed. To explore the mechanisms of checkpoint enforcement, we previously screened for mutants of Saccharomyces cerevisiae which are unable to recover from a transient treatment with a benzimidazole-related microtubule inhibitor because they fail to inhibit subsequent cell cycle steps. Two of the identified genes, BUB2 and BUB3, have been cloned and described (M. A. Hoyt, L. Totis, and B. T. Roberts, Cell 66:507-517, 1991). Here we present the characterization of the BUB1 gene and its product. Genetic evidence was obtained suggesting that Bub1 and Bub3 are mutually dependent for function, and immunoprecipitation experiments demonstrated a physical association between the two. Sequence analysis of BUB1 revealed a domain with similarity to protein kinases. In vitro experiments confirmed that Bub1 possesses kinase activity; Bub1 was able to autophosphorylate and to catalyze phosphorylation of Bub3. In addition, overproduced Bub1 was found to localize to the cell nucleus.
正常细胞增殖要求有丝分裂事件以精心安排的方式发生。细胞利用关卡来阻止细胞周期进程，直到某些先决步骤完成。为了探究关卡执行的机制，我们之前筛选了酿酒酵母的突变体，这些突变体在用苯并咪唑相关的微管抑制剂进行短暂处理后无法恢复，因为它们无法抑制后续的细胞周期步骤。已克隆并描述了两个已鉴定的基因BUB2和BUB3（M. A. 霍伊特、L. 托蒂斯和B. T. 罗伯茨，《细胞》66:507 - 517, 1991）。在此我们展示了BUB1基因及其产物的特征。获得的遗传证据表明Bub1和Bub3在功能上相互依赖，免疫沉淀实验证明了两者之间存在物理关联。BUB1的序列分析揭示了一个与蛋白激酶相似的结构域。体外实验证实Bub1具有激酶活性；Bub1能够自磷酸化并催化Bub3的磷酸化。此外，发现过量表达的Bub1定位于细胞核。
