Control of Ribosomal Gene Transcription in Plants

我实验室头三年的研究目标是将拟南芥建立为一个有用的系统，用于核糖体基因调控的生化和遗传分析。核糖体RNA基因占拟南芥基因组的8%。我们已经从拟南芥基因组文库中分离出大量的核糖体RNA基因，并正在寻找映射到潜在调节区中的基因间隔区的RFLP。我们已经通过S1作图初步确定了转录起始点，并注意到在基因间隔区上游有一个重复的起始点序列。未来的工作将确定这些重复的序列是否代表间隔区启动子，这种间隔区启动子从未在植物中描述过，但在许多其他生物体中存在。我们还将寻找转录本3‘末端形成的位置，以帮助定位拟南芥间隔区中潜在的终止子序列。为了测试潜在的顺式作用元件，如启动子和增强子的活性，我们将构建核糖体微基因，其转录产物在瞬时表达分析中可以与内源核糖体基因转录产物区分开来。一旦测试得到优化，我们将尝试确定间隔区重复序列是否像非洲爪哇和小鼠那样增强转录。我们还将研究生长素对RNA聚合酶I特异性启动的影响。最后，我们将为脊椎动物RNA聚合酶I转录因子UBF的植物同源基因筛选拟南芥基因组和cDNA文库。这些研究旨在建立拟南芥与其他更好物种的核糖体基因组织和生物化学之间的关系，作为可以在植物中进行遗传分析的前奏。

The goal for the first three years' research in my lab is to establish Arabidopsis as a useful system for the biochemical and genetic analysis of ribosomal gene regulation. Ribosomal RNA genes comprise 8% of the total Arabidopsis genome. We have isolated numerous ribosomal RNA genes from an Arabidopsis thaliana genomic library, and are looking for RFLP's that map to the intergenic spacer in potential regulatory regions. We have putatively identified the transcription start site by S1 mapping, and have noticed a duplication of the start site sequence further upstream in the intergenic spacer. Future work will determine if these duplicated sequences represent spacer promoters, which have never been described in plants but are present in many other organisms. We will also look for sites of transcript 3' end formation to help localize potential terminator sequences in the arabidopsis spacer. To test the activity of potential cis-acting elements, such as promoters and enhancers, we will construct ribosomal minigenes whose transcripts can be distinguished from the endogenous ribosomal gene transcripts in transient expression assays. Once the assay is optimized, we will attempt to determine if spacer repeat sequences enhance transcription, as in Xenopus and mouse. We will also examine the effects of auxin on specific initiation by RNA polymerase I. Finally, we will screen Arabidopsis genomic and cDNA libraries for the plant homolog of the vertebrate RNA polymerase I transcription factor, UBF. These studies are aimed at establishing the relationship between ribosomal gene organization and biochemistry in Arabidopsis with other, better species as a prelude to genetic analyses that can be undertaken in plants.***//