Musters W, Venema J, van der Linden G, van Heerikhuizen H, Klootwijk J, Planta R J
Biochemisch Laboratorium, Vrije Universiteit, Amsterdam, The Netherlands.
Mol Cell Biol. 1989 Feb;9(2):551-9. doi: 10.1128/mcb.9.2.551-559.1989.
To develop a system for the analysis of eucaryotic ribosomal DNA (rDNA) mutations, we cloned a complete, transcriptionally active rDNA unit from the yeast Saccharomyces cerevisiae on a centromere-containing yeast plasmid. To distinguish the plasmid-derived ribosomal transcripts from those encoded by the rDNA locus, we inserted a tag of 18 base pairs within the first expansion segment of domain I of the 26S rRNA gene. We demonstrate that this insertion behaves as a neutral mutation since tagged 26S rRNA is normally processed and assembled into functional ribosomal subunits. This system allows us to study the effect of subsequent mutations within the tagged rDNA unit on the biosynthesis and function of the rRNA. As a first application, we wanted to ascertain whether the assembly of a 60S subunit is dependent on the presence in cis of an intact 17S rRNA gene. We found that a deletion of two-thirds of the 17S rRNA gene has no effect on the accumulation of active 60S subunits derived from the same operon. On the other hand, deletions within the second domain of the 26S rRNA gene completely abolished the accumulation of mature 26S rRNA.
为了开发一种用于分析真核生物核糖体DNA（rDNA）突变的系统，我们在一个含着丝粒的酵母质粒上克隆了来自酿酒酵母的完整、具有转录活性的rDNA单元。为了区分质粒衍生的核糖体转录本与rDNA基因座编码的转录本，我们在26S rRNA基因第一结构域的第一个扩展片段内插入了一个18个碱基对的标签。我们证明这种插入表现为中性突变，因为带标签的26S rRNA通常会被加工并组装成功能性核糖体亚基。这个系统使我们能够研究带标签的rDNA单元内后续突变对rRNA生物合成和功能的影响。作为首次应用，我们想确定60S亚基的组装是否依赖于顺式存在的完整17S rRNA基因。我们发现，17S rRNA基因三分之二的缺失对来自同一操纵子的活性60S亚基的积累没有影响。另一方面，26S rRNA基因第二结构域内的缺失完全消除了成熟26S rRNA的积累。
