Wai H H, Vu L, Oakes M, Nomura M
Department of Biological Chemistry, University of California at Irvine, Irvine, CA 92697-1700, USA.
Nucleic Acids Res. 2000 Sep 15;28(18):3524-34. doi: 10.1093/nar/28.18.3524.
Strains of Saccharomyces cerevisiae were constructed in which chromosomal rDNA repeats are completely deleted and their growth is supported by a plasmid carrying a single rDNA repeat, either a plasmid carrying the 35S rRNA gene transcribed from the native promoter by RNA polymerase I or a plasmid carrying the 35S rRNA gene fused to the GAL7 promoter for transcription by RNA polymerase II. This system has made it possible to assess the expression of rDNA by measuring the ability of synthesized rRNA to support cell growth as well as by measuring the actual rRNA synthesized rather than by the use of reporter mini-rDNA genes. Using this system, deletion analysis of the rDNA promoter confirmed the presence of two elements, the upstream element and the core promoter, and showed that basal transcription from the core promoter, if it takes place in vivo as was observed in vitro, is not sufficient to allow cell growth. We have also succeeded in integration of a rDNA repeat and its copy number expansion at the original chromosomal locus, which will allow future mutational analysis not only of rRNA but also other DNA elements involved in rRNA transcription, rDNA replication and recombination within a repeated rDNA structure.
构建了酿酒酵母菌株，其中染色体rDNA重复序列被完全删除，其生长由携带单个rDNA重复序列的质粒支持，该质粒要么携带由RNA聚合酶I从天然启动子转录的35S rRNA基因，要么携带与GAL7启动子融合以由RNA聚合酶II转录的35S rRNA基因。该系统使得通过测量合成的rRNA支持细胞生长的能力以及通过测量实际合成的rRNA来评估rDNA的表达成为可能，而不是通过使用报告小rDNA基因。使用该系统，对rDNA启动子的缺失分析证实了存在两个元件，即上游元件和核心启动子，并表明如果核心启动子在体内发生基础转录（如在体外观察到的那样），则不足以支持细胞生长。我们还成功地在原始染色体位点整合了一个rDNA重复序列并实现了其拷贝数扩增，这将允许未来不仅对rRNA而且对参与rRNA转录、rDNA复制和重复rDNA结构内重组的其他DNA元件进行突变分析。
