Circular permutation of a synthetic eukaryotic chromosome with the telomerator.
Chromosome engineering is a major focus in the fields of systems biology, genetics, synthetic biology, and the functional analysis of genomes. Here, we describe the "telomerator," a new synthetic biology device for use in Saccharomyces cerevisiae. The telomerator is designed to inducibly convert circular DNA molecules into mitotically stable, linear chromosomes replete with functional telomeres in vivo. The telomerator cassette encodes convergent yeast telomere seed sequences flanking the I-SceI homing endonuclease recognition site in the center of an intron artificially transplanted into the URA3 selectable/counterselectable auxotrophic marker. We show that inducible expression of the homing endonuclease efficiently generates linear molecules, identified by using a simple plate-based screening method. To showcase its functionality and utility, we use the telomerator to circularly permute a synthetic yeast chromosome originally constructed as a circular molecule, synIXR, to generate 51 linear variants. Many of the derived linear chromosomes confer unexpected phenotypic properties. This finding indicates that the telomerator offers a new way to study the effects of gene placement on chromosomes (i.e., telomere proximity). However, that the majority of synIXR linear derivatives support viability highlights inherent tolerance of S. cerevisiae to changes in gene order and overall chromosome structure. The telomerator serves as an important tool to construct artificial linear chromosomes in yeast; the concept can be extended to other eukaryotes.
Department of Biochemistry and Molecular Pharmacology and Institute for Systems Genetics, New York University Langone School of Medicine, New York, NY 10016.
Leslie A
LA
Mitchell
Department of Biochemistry and Molecular Pharmacology and Institute for Systems Genetics, New York University Langone School of Medicine, New York, NY 10016 jef.boeke@nyumc.org.
Jef D
JD
Boeke
D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D018244:Chromosomes, Artificial, Yeast / Q000235:genetics*; D004270:DNA, Circular / Q000235:genetics*; D015252:Deoxyribonucleases, Type II Site-Specific / Q000235:genetics / Q000378:metabolism; D016521:Electrophoresis, Gel, Pulsed-Field; D015966:Gene Expression Regulation, Fungal; D008969:Molecular Sequence Data; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism; D012689:Sequence Homology, Nucleic Acid; D058615:Synthetic Biology / Q000379:methods*; D016615:Telomere / Q000235:genetics*
D004270:DNA, Circular; D029701:Saccharomyces cerevisiae Proteins; C554167:URA3 protein, S cerevisiae; C037725:SCEI protein, S cerevisiae; D015252:Deoxyribonucleases, Type II Site-Specific
Sc2.0; chromosome engineering; synthetic chromosome; telomerator; telomeric silencing