Abstract
BackgroundSynthetic 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.ResultsWe present a modified chromosome assembly technique in which eukaryotic chromosomes as large as ~500 kb can be assembled from cloned ~100 kb fragments. We used this technique to clone fragments spanning P. tricornutum chromosomes 25 and 26 and to assemble these fragments into single, chromosome-sized molecules. We found that addition of yeast replication origins improved the cloning, assembly, and maintenance of the large chromosomes in yeast. Furthermore, purification of the fragments to be assembled by electroelution greatly increased assembly efficiency.ConclusionsEntire eukaryotic chromosomes can be successfully cloned, maintained, and manipulated in yeast. These results highlight the improvement in assembly and maintenance afforded by including yeast replication origins in eukaryotic DNA with moderate G + C content (48%). They also highlight the increased efficiency of assembly that can be achieved by purifying fragments before assembly.
Highlights
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
These scaffolds are described as chromosomes in reports on the genome sequence [20], ongoing work to completely close the genome sequence may collapse some of these scaffolds without telomere-to-telomere coverage into single chromosomes
We chose P. tricornutum chromosomes 25 and 26 as targets to assemble entire eukaryotic chromosomes in yeast because they are the smallest chromosomes for which telomere-to-telomere assembly exists
Summary
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. 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 improves the yeast-based cloning of large (>150 kb) eukaryotic DNA with moderate G + C content. Synthetic biology can be described as the iterative process of modeling biological phenomena, building genetic devices to implement these models, and evaluating device performance This discipline practiced at the scale of entire chromosomes can be defined as synthetic genomics. We have previously used the synthetic genomic approach with prokaryotic genomes which are typically small, circular, and contained on a single molecule These qualities allow for efficient maintenance and manipulation of the chromosome in yeast [7,8,9,10]. Chromosomes from eukaryotic organelles have been synthesized or assembled in yeast including the mitochondrion [11] and the chloroplast [12], but to our knowledge, no entire eukaryotic nuclear chromosome has been cloned in yeast
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
