Assembly of eukaryotic algal chromosomes in yeast

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.

Diatom centromeres suggest a mechanism for nuclear DNA acquisition

“…We hypothesized that a centromeric region of a diatom chromosome would support maintenance of a nuclear episome, as this is a useful experimental method of confirming centromere function for other organisms (32,43). To identify a diatom centromeric region, we first examined the shortest P. tricornutum chromosomes with telomere-to-telomere assembly (25 and 26) (39), which were each previously cloned as five overlapping ∼100-kb (76.6-142.6 kb) DNA fragments (44). In our prior studies (37,38), sequences supporting episome maintenance in P. tricornutum were characterized by greatly improved ex-conjugant colony yield compared with plasmids incapable of episome maintenance.…”

Nitrate Reductase Knockout Uncouples Nitrate Transport from Nitrate Assimilation and Drives Repartitioning of Carbon Flux in a Model Pennate Diatom

“…The plasmid was constructed to contain yeast replication origins and endogenous P. tricornutum NR fused to YFP under the control of the native NRprom and NRterm, as previously described (Karas et al, 2013). The conjugation produced numerous colonies that grew on NH 4 + -amended SW-agar plates under Zeocin selection.…”

Bacterial genome reduction using the progressive clustering of deletions via yeast sexual cycling

“…Cloning of a whole genome in yeast has been demonstrated for genomes from several bacteria (Gibson et al 2008Lartigue et al 2009;Benders et al 2010;Karas et al 2011Karas et al , 2013aTagwerker et al 2012). This approach has also been expanded to eukaryotic chromosomes (Karas et al 2013b). The procedure for transferring bacterial genomes into yeast has been further facilitated by our discovery of genome transfer within a mixture of bacterial cells and yeast spheroplasts (Karas et al 2013a).…”