Dissection of centromere and associated replication origins

着丝粒和相关复制起点的解剖

• 批准号: 09044236
• 负责人: MATSUOKA Masayoshi
• 金额: $ 2.18万
• 依托单位: Kumamoto Institute of Technology
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for international Scientific Research
• 财政年份: 1997
• 资助国家: 日本
• 起止时间: 1997 至 1998
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09044236/
• 关键词: centromere; Yarrowia lipolytica; autonomous replication; plasmids; transformation; nuclear matrix; site-directed mutagenesis; 複製起点; 染色体

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.

在解脂耶氏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，并显示其包含与着丝粒相关的复制起点。这些片段含有与解脂 Y 中 6 条染色体中的 CEN1、CEN3 和 CEN5 密切相关的复制起点，如下实验所示： 1）这些克隆在脉冲场凝胶电泳中与不同染色体条带杂交； 2) 当含有CEN的片段整合到染色体上的异位位点时，观察到双着丝粒染色体发生染色体断裂； 3) 着丝粒质粒可用于克隆来自多个基因组位置的许多复制起点。通过 2-D 凝胶电泳检测复制 DNA 分叉。因此，只有在复制起点和着丝粒同时存在的情况下，解脂耶氏酵母的转化才是可行的，后者对于核质粒的正确分配或复制子质粒 DNA 的解析是必需的。然而，与核基质的简单结合不足以实现 CEN 功能。最小的 CEN1 区域限制在约 130 bp 内。在含起点的载体上对基因组 DNA 进行鸟枪式克隆，通过高频转化发现了三个额外的克隆。这些新克隆是 CEN2、CEN4 和位于最小 CEN1 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。通过定点诱变研究了 CEN 区的保守序列元件。