Dissection of centromere and associated replication origins

在解脂耶氏酵母中获得了三个在质粒上显示重复复制（ARS）活性的DNA片段，并显示其具有与着丝粒相关的复制起点。结果表明：1）在脉冲场凝胶电泳中，这些克隆与染色体上不同的带型杂交，2）当含CEN的片段整合到染色体上的异位位点时，双着丝粒染色体出现断裂; 3）着丝粒质粒可用于克隆来自几个基因组位置的许多复制起点。因此，只有在同时存在复制起点和着丝粒的情况下，解脂耶氏酵母的转化才是可行的，而着丝粒是正确分配核质粒或解析复制子质粒DNA所必需的。然而，仅与核基质结合并不足以实现CEN的功能，CEN 1的最小区域被限制在约130 bp内。在含有来源的载体上的基因组DNA的鸟枪克隆揭示了通过高频转化的另外三个克隆。这些新克隆是CEN 2、CEN 4和与最小CEN 1侧翼但不重叠的片段。如果我们假设解脂耶氏酵母着丝粒是在粟酒裂殖酵母或高等真核生物中观察到的区域结构，则可以解释这一发现。通过定点突变的方法对CEN区的保守序列元件进行了研究。

Three DNA fragments displaying autonomously-replicating (ARS) activity on plasmids in the yeast Yarrowia lipolytica have been obtained and shown to harbor replication origins associated with centromeres. These fragments contain replication origins closely associated with CEN1, CEN3 and CEN5 among six chromosomes in Y lipolytica as shown by the following experiments : 1) These clones hybridized with different chromosomal bands in the pulse-field gel electrophoresis ; 2) When the CEN-containing fragments were integrated in the ectopic sites on chromosome, chromosome breakage was observed for the dicentric chromosomes ; 3) a centromeric plasmid can be used to clone many replication origins coming from several genomic locations. The replicating DNA fork was detected by 2-D gel electrophoresis.Transformation of Y.lipolytica is therefore feasible only in the simultaneous presence of a replication origin and a centromere, the latter being necessary either for a proper partition of nuclear plasmids or for a resolution of replicated daughter plasmid DNAs. However, a simple binding to a nuclear matrix was not sufficient for a CEN function.The minimal CEN1 region was confined within about 130 bp. A shot-gun cloning of genomic DNA on an origin-containing vector revealed three additional clones via high-frequency transformation. These new clones were CEN2, CEN4 and a fragment flanking but not overlapping with minimal CEN1. This finding could be explained if we assume that Y.lipolytica centromeres are regional structures as observed in Schizosaccharomyces pombe or higher eukaryotes. The conserved sequence elements in CEN region are investigated by means of site-directed mutagenesis.