Consequences of a telomerase-related fitness defect and chromosome substitution technology in yeast synIX strains
Laura H. McCulloch, Vijayan Sambasivam, Amanda L. Hughes, Narayana Annaluru, Sivaprakash Ramalingam, Viola Fanfani, Evgenii Lobzaev, Leslie A. Mitchell, Jitong Cai, Breeana G. Anderson, Abena Apaw, Pavlo Bohutskyi, Erin Buchanan, Daniel T. Chang, M. Chen, Eric M. Cooper, Amanda Deliere, Kallie Drakos, Justin T. Dubin, Christopher Fernandez, Zheyuan Guo, Thomas F. Harrelson, Dongwon Lee, Jessica E. McDade, Scott Melamed, Héloïse Muller, Adithya Murali, José U. Niño Rivera, Mira C. Patel, Mary Rodley, Jenna Schwarz, Nirav Shelat, Josh S. Sims, Barrett Steinberg, James J. Steinhardt, Rishi Trivedi, Christopher Von Dollen, Tianyi Wang, Remus Wong, Yijie Xu, Noah Young, Karen Zeller, Allen Zhang, Hua Jiang, John LaCava, Martin S. Taylor, William R. Bishai, Giovanni Stracquadanio, Lars M. Steinmetz, Joel S. Bader, Weimin Zhang, Jef D. Boeke, Srinivasan Chandrasegaran
Abstract
We describe the complete synthesis, assembly, debugging, and characterization of a synthetic 404,963 bp chromosome, synIX (synthetic chromosome IX). Combined chromosome construction methods were used to synthesize and integrate its left arm (synIXL) into a strain containing previously described synIXR. We identified and resolved a bug affecting expression of EST3, a crucial gene for telomerase function, producing a synIX strain with near wild-type fitness. To facilitate future synthetic chromosome consolidation and increase flexibility of chromosome transfer between distinct strains, we combined chromoduction, a method to transfer a whole chromosome between two strains, with conditional centromere destabilization to substitute a chromosome of interest for its native counterpart. Both steps of this chromosome substitution method were efficient. We observed that wild-type II tended to co-transfer with synIX and was co-destabilized with wild-type IX, suggesting a potential gene dosage compensation relationship between these chromosomes.