Control of Ribosomal Gene Transcription in Plants

我的实验室前三年的研究目标是建立拟南芥作为核糖体基因调控的生化和遗传分析的有用系统。核糖体RNA基因占拟南芥基因组总数的8%。我们已经从拟南芥基因组文库中分离了许多核糖体RNA基因，并正在寻找潜在调控区域的基因间间隔区RFLP。我们已经通过S1定位推测出了转录起始位点，并注意到在更远的上游基因间隔中有一个重复的起始位点序列。未来的工作将确定这些重复序列是否代表间隔启动子，它们从未在植物中被描述过，但存在于许多其他生物中。我们还将寻找转录本3'末端形成的位点，以帮助定位拟南芥间隔段中潜在的终止序列。为了测试潜在的顺式作用元件（如启动子和增强子）的活性，我们将构建核糖体小基因，其转录物可以在瞬时表达试验中与内源性核糖体基因转录物区分。一旦实验得到优化，我们将尝试确定间隔重复序列是否能增强转录，就像在非洲爪蟾和小鼠中一样。我们还将研究生长素对RNA聚合酶I特异性起始的影响。最后，我们将筛选拟南芥基因组和cDNA文库，以获得脊椎动物RNA聚合酶I转录因子UBF的植物同源物。这些研究旨在建立拟南芥核糖体基因组织和生物化学与其他更好的物种之间的关系，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.***//