Hegemann J H, Shero J H, Cottarel G, Philippsen P, Hieter P
Institut fuer Mikrobiologie und Molekularbiologie, Justus Liebig Universitaet, Giessen, Federal Republic of Germany.
Mol Cell Biol. 1988 Jun;8(6):2523-35. doi: 10.1128/mcb.8.6.2523-2535.1988.
Saccharomyces cerevisiae centromeres have a characteristic 120-base-pair region consisting of three distinct centromere DNA sequence elements (CDEI, CDEII, and CDEIII). We have generated a series of 26 CEN mutations in vitro (including 22 point mutations, 3 insertions, and 1 deletion) and tested their effects on mitotic chromosome segregation by using a new vector system. The yeast transformation vector pYCF5 was constructed to introduce wild-type and mutant CEN DNAs onto large, linear chromosome fragments which are mitotically stable and nonessential. Six point mutations in CDEI show increased rates of chromosome loss events per cell division of 2- to 10-fold. Twenty mutations in CDEIII exhibit chromosome loss rates that vary from wild type (10(-4)) to nonfunctional (greater than 10(-1)). These results directly identify nucleotides within CDEI and CDEIII that are required for the specification of a functional centromere and show that the degree of conservation of an individual base does not necessarily reflect its importance in mitotic CEN function.
酿酒酵母着丝粒有一个由三个不同的着丝粒DNA序列元件（CDEI、CDEII和CDEIII）组成的特征性120碱基对区域。我们在体外产生了一系列26个CEN突变（包括22个点突变、3个插入和1个缺失），并通过使用一种新的载体系统测试了它们对有丝分裂染色体分离的影响。构建酵母转化载体pYCF5，将野生型和突变型CEN DNA导入到有丝分裂稳定且非必需的大型线性染色体片段上。CDEI中的六个点突变显示，每个细胞分裂的染色体丢失事件发生率增加了2至10倍。CDEIII中的20个突变表现出从野生型（10^(-4)）到无功能（大于10^(-1)）不等的染色体丢失率。这些结果直接确定了CDEI和CDEIII中对于功能性着丝粒的指定所必需的核苷酸，并表明单个碱基的保守程度不一定反映其在有丝分裂CEN功能中的重要性。
