Dissection of centromere and associated replication origins

在解脂雅罗维菌的酵母中获得了三个在质粒上显示自主复制(ARS)活性的DNA片段，并证明它们含有与着丝粒相关的复制起点。实验表明：1)这些克隆在脉冲场凝胶电泳中与不同的染色体带杂交；2)当含有CEN的片段整合到染色体上的异位位置时，观察到双着丝粒染色体的染色体断裂；3)着丝粒质粒可用于克隆来自多个基因组位置的多个复制起点。因此，只有在复制起点和着丝粒同时存在的情况下，才能转化解脂耶尔森菌，而着丝粒是正确划分核质粒或分离复制的子质粒DNA所必需的。然而，对于CEN功能来说，简单地与核基质结合是不够的，最小的CEN1区域被限制在大约130个核苷酸以内。在含有起源的载体上进行的基因组DNA的枪击克隆发现，通过高频转化，又有三个克隆。这些新克隆是CEN2、CEN4和一个位于侧翼但不与最小CEN1重叠的片段。如果我们假设解脂耶尔森菌着丝粒是区域结构，就可以解释这一发现，就像在裂殖酵母或高等真核生物中观察到的那样。用定点突变的方法研究了CEN区的保守序列元件。

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.