Much of the eukaryotic transcriptional machinery is conserved from yeast to human. However, the distance over which transcriptional activation can occur differs between Saccharomyces cerevisiae and metazoans. In S. cerevisiae, the upstream activating sequence (UAS) is generally found within 300 base pairs of the transcription start site; when the UAS is moved too far away, activation no longer occurs. In contrast, metazoan enhancers can activate from as far as 100 kilobases from the start site. In past work, our lab identified five genes that, when mutant, allow transcription activation to occur at a greater-than-normal distance from the GAL1 UAS. As this long-distance activation phenotype was weak, we have now studied long-distance activation as a polygenic trait, isolating strains with multiple mutations that together confer a strong phenotype. To do this, we constructed strains containing two reporters, HIS3 and URA3. For each reporter, the GAL1 UAS was placed approximately 800 base pairs upstream of the transcription start sites. By iterative selection for stronger and stronger expression of HIS3, followed by screening for stronger expression of URA3, we isolated three strains, each containing multiple mutations that contribute to the strength of the long distance activation phenotype. Causative mutations were identified in MOT3, GRR1, MIT1, PTR3, YOR019W, and MSN2 that contribute to the long distance activation phenotype. Strains containing multiple mutations were found to activate the reporter construct at distances up to 2 kilobases. Microarray analysis revealed genome wide transcriptional changes in the mutant strains. Statistical analysis of the microarray results suggests other potential sites of long distance activation throughout out the genome. These results have extended our understanding of mutations that allow long distance activation and have demonstrated the value of studying a phenotype as a polygenic trait.
| Identifer | oai:union.ndltd.org:harvard.edu/oai:dash.harvard.edu:1/11169767 |
| Date | 14 October 2013 |
| Creators | Reavey, Caitlin Teresa |
| Contributors | Winston, Fred Marshall |
| Publisher | Harvard University |
| Source Sets | Harvard University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis or Dissertation |
| Rights | open |
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