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.***//
