Circular permutation of a synthetic eukaryotic chromosome with the telomerator

Leslie A. Mitchell, Jef D. Boeke

Research output: Contribution to journalArticle

Abstract

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.

Original languageEnglish (US)
Pages (from-to)17003-17010
Number of pages8
JournalProceedings of the National Academy of Sciences of the United States of America
Volume111
Issue number48
DOIs
Publication statusPublished - Dec 2 2014
Externally publishedYes

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Keywords

  • chromosome engineering
  • Sc2.0
  • synthetic chromosome
  • telomerator
  • telomeric silencing

ASJC Scopus subject areas

  • General

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