Centromeres in many species are found to reside near origins of replication that are activated early in S-phase, yet the biological significance of this conserved early replication of centromeres is unknown. Previous research has shown that the activation time of an origin is affected by its chromosomal location. A recent study has found that centromeres in the yeast Saccharomyces cerevisiae advance the activation time of nearby origins, and that this effect depends on the centromere’s ability to establish a functional kinetochore. These observations lead to the hypothesis that centromeres need to be replicated early to allow sufficient time for the assembly of functional kinetochores. If kinetochores fail to be established in a timely fashion, chromosomes may not be correctly bi-orient at mitosis. Daughter cells therefore may inherit the incorrect number of chromosomes creating conditions of aneuploidy such as those found in Down syndrome and many cancers. I will test the importance of early centromere replication by deleting origins immediately flanking a yeast centromere to delay that centromere’s replication. I will then conduct an assay to test for the rate of chromosome mis-segregation events in the mutant strain compared to the wild-type strain. I anticipate that the mutant strain may have a higher rate of loss for the modified chromosome, suggesting that early replication of centromeres is important for maintaining genome stability. This project will also provide insights into the regulation of the temporal program of replication origin activation and provide the first functional test for the consequences of disrupting the temporal program of chromosome replication.

Sequencing the genome of an organism, or determining the order of the base pairs in its DNA, is one approach to understanding that organism’s chromosome structure.  Genome sequence assembly is accomplished with the help of computer programs, which often fail to include or map repetitious sequences in the final chromosome assembly.  One such unmapped sequence in the Lachancea waltii (L. waltii) yeast genome is the ribosomal DNA (rDNA), a sequence composed of hundreds of identical repeating segments that encodes the RNA component of ribosomes, the protein-producing machines in the cell.  My previous research has shown the rDNA is located in two large sequence gaps on chromosome 8, the only chromosome published incompletely as three separate segments of sequence.  My current research involves determining the order and orientation of these sequence scaffolds to verify and complete the assembly of L. waltii chromosome 8.  To investigate the arrangement of the three scaffolds, I am first performing indirect end labeling, where I am cleaving the chromosome with restriction enzymes to produce a specific fragmentation pattern.  Alignment of this pattern with that expected based on the proposed structure of chromosome 8 would validate the proposed chromosome structure and rDNA location.  In addition, I am performing a snap-back assay, a test that will indicate the orientation of the rDNA segment repeats – thereby verifying if the rDNA is present in one or two locations.  Finally, I am degrading the ends of chromosome 8 using an exonuclease – an end-specific enzyme – to determine the sequences, and thus the identity and orientation of the scaffolds, that are present at the chromosome ends.  By generating a complete, contiguous sequence of L. waltii chromosome 8, we hope to provide a more in-depth profile of this relatively uncharacterized yeast for future studies on chromosome evolution.