Assembly of eukaryotic algal chromosomes in yeast

Journal of Biological Engineering

Philip D Weyman¹  J Craig Venter²  Hamilton O Smith¹  Clyde A Hutchison¹  John I Glass²  Andrew E Allen³  Ray-Yuan Chuang²  Vladimir N Noskov²  Isaac T Yonemoto¹  Christian Tagwerker¹  Christopher L Dupont³  Ying-Chi Lin¹  Wolfgang J Hermann¹  Jelena Jablanovic¹  Bhuvan Molparia¹  Bogumil J Karas¹ 

[1] Department of Synthetic Biology and Bioenergy, J. Craig Venter Institute, 10355 Science Center Dr., San Diego, CA 92121, USA;Department of Synthetic Biology and Bioenergy, J. Craig Venter Institute, 9704 Medical Center Dr., Rockville, MD 20850, USA;Department of Microbial and Environmental Genomics, J. Craig Venter Institute, 10355 Science Center Dr., San Diego, CA 92121, USA

关键词: Phaeodactylum tricornutum;    Autonomously replicating sequence (ARS);    Saccharomyces cerevisiae;    TAR cloning;    Chromosome;    Eukaryote;   

Others  :  804998
DOI  :  10.1186/1754-1611-7-30

 received in 2013-09-13, accepted in 2013-11-27,  发布年份 2013

【 摘 要 】

Background

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.

Results

We 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.

Conclusions

Entire 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.

【 授权许可】

   
2013 Karas et al.; licensee BioMed Central Ltd.

【 图 表 】

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