Precise, complete and timely replication of eukaryotic genomes is a prerequisite to cell division. Each chromosome replicates in a defined temporal order that is dictated by the variable activation timings and efficiencies of replication origins. However, so far the mechanisms regulating origin activity have remained elusive. Replication origins are best understood in the budding yeast Saccharomyces cerevisiae. Powerful comparative genomic approaches are possible in budding yeasts due to the evolutionary range of sequenced genomes available and their tractability to genetic approaches. Previously, functional sequence elements at replication origins have been identified based upon their phylogenetic sequence conservation amongst closely related species of the sensu stricto group. To gain insight into the selective pressures contributing to this phylogenetic conservation, mutant strains with chromosomally inactivated origins were grown in competition with wild-type strains. Origin mutant strains did not have a growth defect compared to the wild-type, suggesting that the selective advantage conferred by evolutionary conserved origins is not the requirement for a rapid cell cycle time. To improve the reference sequence annotation, the S. cerevisiae genome was systematically screened for origin function, confirming more than 200 additional replication origins. The resulting comprehensive map of origin locations in S. cerevisiae was used to assess the accuracy of origin predictions from published studies and two newly developed techniques. These two approaches use high-throughput sequencing to either identify replication origin locations or measure replication dynamics genome-wide. Using the latter method on haploid and diploid S. cerevisiae strains showed that replication dynamics are independent of cell ploidy. Genome replication in divergent budding yeasts was investigated using a combination of replication timing profiles acquired with deep sequencing and plasmid-based assays. These analyses formed the basis for a comparative genomics approach, which revealed that the relative order of genome replication is conserved. A minority of replication origins with identical genomic locations show differences in activity between the analyzed species. To gain insight into the mechanisms underlying origin regulation, the replication dynamics of a hybrid between S. cerevisiae and its most distant relative in the sensu stricto group - S. bayanus - were measured. Replication origin function was found to be controlled by both local and global regulators .
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:603592 |
| Date | January 2012 |
| Creators | Muller, Carolin Anne |
| Publisher | University of Nottingham |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
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