| Saccharomyces cerevisiae is an excellent model organism and its genome is the best characterized of any eukaryote. To extend our understanding of transcriptional units in S. cerevisiae, we have carried out both computational and experimental analysis to discover and characterize novel genomic elements in the S. cerevisiae genome. One minimally addressed area in S. cerevisiae research is the mapping of transcription start sites (TSS). Mapping of TSS in S. cerevisiae has the potential to contribute to our understanding of gene regulation, transcription, mRNA stability, and other aspects of RNA biology. We have developed and applied a high throughput approach, 5' SAGE, to map 5 ' TSS in S. cerevisiae. We have identified TSS for 2231 (38.6%) of S. cerevisiae genes. TSS identified in this study are consistent with published results, as well as with primer extension results described here, and are consistent with expectations based on previous work on transcription initiation. From these results, we have identified a conserved motif characteristic of the TSS consensus. Combined with comparative methods, the TSS data made possible the identification of a previously unrecognized gene, uncovered errors in previous annotation, and identified potential regulatory RNAs and upstream ORFs in 5' UTR.; Using another approach, we have taken advantage of ten recently sequenced hemiascomycete fungal genomes to computationally identify additional elements. Fifteen new uORF containing genes, five new introns with two located in the UTR region, one new gene, and four sequencing errors are verified in S. cerevisiae by 5' RACE, primer extension or Northern blot. The newly discovered uORFs from seven genes have been characterized using a Luciferase reporter system to identify their potential role in gene expression. Preliminary results suggest that most of these uORFs attenuate gene expression. Experimental investigation of the role of UTR intron has been carried out using COX4 as a model and has showed this intron does not measurably affect gene expression.; Together, these results improve our understanding of the S. cerevisiae genome, the set of genes in this organism, and how these genes are regulated. |
