Creating a Circular Chromosome in the Budding Yeast Saccharomyces cerevisiae Ethan Chandler and Melissa Mefford Morehead State University Department of Biology and Chemistry, Morehead KY 40351

Expected Results & Introduction Methodology Future Plans

At the ends of linear chromosomes DNA cassettes will be constructed • Strains with a circular chromosome XI are expected to have normal there are unique regions of repeated that have homology to the sub- Selectable Marker Selectable Marker nucleotide sequences called telomeres. telomeric regions of chromosome fitness as compared to strains with In each round of DNA replication, the XI. In one cassette, a selectable only linear chromosomes. termini of telomeres may shorten. To auxotrophic marker, HIS3, and half • The overall fecundity of strains maintain telomeres an enzyme called of the URA3 gene will be added; with a circular chromosome XI is telomerase adds nucleotides at the ends the cassette for the other end will expected to be reduced due to of the chromosomes. The function of contain the complimentary half of difficulties with crossing over in telomerase in telomere maintenance has URA3 and the other auxotrophic meiosis. important implications for human marker, LEU2. These cassettes will • If the circular chromosomes are health. Lack of telomerase function and be transformed into yeast, where viable, then a strain will be telomere shortening plays a role in they can integrate into the sub- genetically engineered to have all aging, while unregulated increased telomeric regions of Chromosome 16 of its chromosomes circularized telomerase activity occurs in most XI. To select for integration, strains in the near future. DNA cassettes human cancers. Given these significant of yeast will be grown on media containing selectable effects on organisms, we will lacking histidine and leucine. Yeast markers are inserted experimentally explore how linear strains that grow on this media will into the ends of chromosomes, telomeres, and then be transferred onto a media Chromosome XI telomerase evolved in the simple model lacking uracil, which will select for Key Literature organism Saccharomyces cerevisiae. To cells that underwent recombination accomplish this, I will genetically to create an intact URA3 gene. The engineer the linear chromosome XI of intact gene will create a circular Klar, A. J., Stratnern, J. N., Hicks, J. B., & Prudente, D. (1983). Efficient Production Saccharomyces cerevisiae into a chromosome. of a Ring Derivative of Chromosome III by circularized version that lacks The viability and fitness of strains the Mating-Type Switching Mechanism in telomeres. with a circular chromosome will Saccharomyces cerevisiae. Molecular and then be characterized. Cellular Biology, 3(5), 803-810. Naito, T., Matsuura, A., & Ishikawa, F. (1998). Circular Chromosome Formation in a Fission Yeast Mutant Defective in two ATM Homologues. Nature Genetics, 20, 203-206.

Acknowledgements The Ends of Chromosome XI: The DNA cassette is inserted between URA3 can recombine and circularize the the start of the telomeric sequence chromosome. (red) and the terminal-most gene Thank you to the KBRIN New Faculty Individual cells of S. cerevisiae Yeast strains with circular chromosomes (YKL 224 and YKR 106) will be selected in media lacking uracil. Start Up Award to Dr. Melissa Mefford Image Credit: https://www2.le.ac.uk for funding. (NIGMS P20GM103436)