Chromosome structure varies from a single circular chromosome in prokaryotes to multiple linear chromosomes in eukaryotes. Linear chromosomes differ in that they possess specialized ends, called telomeres, that play important roles in protecting the ends from degradation. Furthermore, telomeres cannot be fully copied during DNA replication without the enzyme complex telomerase. Since telomerase is not expressed in all cells, telomeres shorten with aging. On the other hand, overexpression of telomerase is observed in gt;85% of human cancers. Given that telomeres create this end-replication problem, our lab is interested in ultimately understanding why linear chromosomes evolved in the first place. To address this broad question, we are taking a novel genetic approach to systematically convert each of the linear chromosomes into circularized versions using the single-celled eukaryote Saccharomyces cerevisiae. Our approach involves building two DNA cassettes with selectable marker elements that are integrated into the left and right arms of each chromosome in a haploid yeast strain. The cassettes are designed so that recombination between the two regions will reconstitute a functional marker gene, allowing genetic selection of circularized chromosomes. To date, we have isolated a circularized version of Ch. IV, the longest of the 16 yeast chromosomes. We have verified recombination between the cassette via PCR across the newly formed junction. The circularized Ch. IV yeast form colonies slightly smaller in size and exhibit a ~10% decrease in doubling time relative to wild-type. Together, these data demonstrate that our approach can generate viable yeast with individual chromosomes circularized. We are currently analyzing cellular morphology using light microscopy, testing the ability of the circularized yeast to undergo mating and sporulation, and analyzing impacts on gene expression. A longer-term goal of the lab is to use this approach to attempt to circularize the other 15 chromosomes and explore how linear chromosomes evolved.
This work was funded by a KY INBRE IDeA grant (NIGMS P20GM103436) to Dr. Melissa Mefford.
