Assembly of eukaryotic algal chromosomes in yeast

Bogumil J Karas, Bhuvan Molparia, Jelena Jablanovic, Wolfgang J Hermann, Ying-Chi Lin, Christopher L Dupont, Christian Tagwerker, Isaac T Yonemoto, Vladimir N Noskov, Ray-Yuan Chuang, Andrew E Allen, John I Glass, Clyde A Hutchison, Hamilton O Smith, J Craig Venter, Philip D Weyman

Synthetic genomic approaches offer unique opportunities to use powerful yeast and Escherichia coli genetic systems to assemble and modify chromosome-sized molecules before returning the modified DNA to the target host. For example, the entire 1 Mb Mycoplasma mycoides chromosome can be stably maintained and manipulated in yeast before being transplanted back into recipient cells. We have previously demonstrated that cloning in yeast of large (> ~ 150 kb), high G + C (55%) prokaryotic DNA fragments was improved by addition of yeast replication origins every ~100 kb. Conversely, low G + C DNA is stable (up to at least 1.8 Mb) without adding supplemental yeast origins. It has not been previously tested whether addition of yeast replication origins similarly improves the yeast-based cloning of large (>150 kb) eukaryotic DNA with moderate G + C content. The model diatom Phaeodactylum tricornutum has an average G + C content of 48% and a 27.4 Mb genome sequence that has been assembled into chromosome-sized scaffolds making it an ideal test case for assembly and maintenance of eukaryotic chromosomes in yeast.