Synthesis and Restructuring of a Yeast Chromosome

基于酿酒酵母的合成形式的设计将用于回答各种各样的深刻的生物学问题，包括与自由生活的真核生物相容的最小基因集以及基因组和染色体稳定性的基本要求。最终，一个全新版本的S。酿酒计划。作为第一步，该项目将合成一个中等大小的染色体，IX，约440 kb长。对天然染色体序列的计划性改变可能是无限的，因此必须对将被并入合成染色体的特定改变给予很多考虑。该项目将使用迭代重组方法，其中约30至100 kb的片段依次替换野生型片段，并且在任何给定阶段对该项目进行的投资范围有限。如果任何特定的片段是不可活的，它可以在生长缺陷的性质被映射和诊断之后被重新合成。待在基因组中缺失或重新定位的特定基因组特征包括端粒区、重复序列如转座子序列、tRNA基因、内含子、沉默区和某些非必需基因。最重要的是，一个内部基因组改组机制将被内置到合成酵母染色体中，并进行测试。这将通过在所有非必需基因的3'UTR中包括对称的loxP位点来实现。将测试这些染色体在极低水平表达的Cre重组酶存在下重组和重排的能力。这一过程将在合成染色体上产生基因内容和顺序不同的“基因组群”。对这些群体的分析将提供关于最小基因集的信息，以及探测潜在基因（或其他特征）邻接规则和其他基因组结构要求。该项目将首次允许深入的问题被问及染色体的基本属性，基因组组织，基因内容，RNA剪接的功能，原核生物和真核生物之间的区别，以及其他许多与进化有关的问题。事实上，完全合成的染色体（最终是完全合成的基因组）的可用性允许直接测试无法以任何其他方式解决的进化问题。最终将被设计和提炼的“合成酵母”很可能在实际应用中发挥重要作用。值得注意的是，酵母是用于工业发酵的杰出生物，具有广泛的实际用途，包括从农产品和副产品生产乙醇。许多教育机会，无论是在约翰霍普金斯大学内外的墙壁将春天从该项目。除了新的课程内容和一个新的整体课程外，还将在一个发展中国家（印度）创建课程内容，并将发起涉及当地有才华的高中学生、高中教师和对该项目有浓厚兴趣的马里兰州科学中心的活动。新课程将侧重于让本科生直接参与大规模功能基因组项目。

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.