Synthesis and Restructuring of a Yeast Chromosome

Johns Hopkins University, Baltimore MD

Investigators

Abstract

The design of a synthetic form based on Saccharomyces cerevisiae will be used to answer a wide variety of profound biological questions, including the minimum gene set compatible with free-living eukaryotic life and the fundamental requirements for genome and chromosome stability. Ultimately, an entirely new version of S. cerevisiae is planned. As a first step a medium sized chromosome, IX, about 440 kb long, will be synthesized in this project. Planned alterations to native chromosome sequences are potentially infinite in number and so much thought must be given to the specific alterations to be incorporated into the synthetic chromosome. This project will use an iterative recombinational approach in which segments of ~30 to 100 kb sequentially replace wild-type segments and with the benefit that the investment made in the project at any given stage is limited in scope. If any particular segment is inviable, it can be resynthesized after the nature of the growth defect is mapped and diagnosed. Specific genomic features to be deleted or relocated in the genome include telomeric regions, repeats such as transposon sequences, tRNA genes, introns, silenced regions, and certain nonessential genes. Most importantly, an internal genome reshuffling mechanism will be built into the synthetic yeast chromosome, and tested. This will be accomplished by including symmetric loxP sites in the 3' UTRs of all nonessential genes. The ability of these chromosomes to recombine and rearrange in the presence of Cre recombinase expressed at very low levels will be tested. This process will generate "genome swarms" differing in gene content and order on the synthetic chromosome. Analysis of these swarms will provide information on minimal gene sets as well as probing underlying gene (or other feature) adjacency rules and other genome structural requirements. This project will allow for the first time, deep questions to be asked about fundamental properties of chromosomes, genome organization, gene content, the function of RNA splicing, the distinction between prokaryotes and eukaryotes, and numerous other questions relating to evolution. In fact the availability of a fully synthetic chromosome (and ultimately a fully synthetic genome) allows for direct testing of evolutionary questions that cannot be addressed in any other way. The "synthetic yeast" that will eventually be designed and refined is likely to play an important role in practical applications. Notably, yeast is the preeminent organism used for industrial fermentations, with a wide variety of practical uses, including ethanol production from agricultural products and by-products. Numerous educational opportunities, both within Johns Hopkins University and outside its walls will spring from the project. In addition to new course content and a new course overall, course content will be created in a developing country (India) and activities involving talented local high school students, high school teachers, and the Maryland Science Center, who have taken a keen interest in the project, will be initiated. The new course will focus on involving undergraduates directly in a large scale functional genomic project.