Synthesis and Restructuring of a Yeast Chromosome

基于酿酒酵母的合成形式的设计将被用来回答广泛的深刻的生物学问题，包括与自由生活的真核生物相适应的最小基因集，以及对基因组和染色体稳定性的基本要求。最终，一个全新版本的酿酒酵母计划问世。作为第一步，这个项目将合成一个中等大小的染色体IX，大约440kb长。对天然染色体序列的有计划的改变可能在数量上是无限的，所以必须对要结合到合成染色体中的特定改变给予很大的考虑。该项目将使用迭代重组方法，其中约30到100 kb的片段依次取代野生型片段，其好处是在任何给定阶段对项目的投资都受到范围的限制。如果任何特定的片段是不可行的，它可以在生长缺陷的性质被绘制和诊断后重新合成。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、沉默区域和某些非必需基因。最重要的是，内部基因组重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‘UTRs中包括对称的loxP位点来实现。这些染色体在低水平表达的Cre重组酶存在的情况下重组和重排的能力将被测试。这一过程将产生“基因组群”，这些基因组在合成染色体上的基因内容和顺序不同。对这些群体的分析将提供关于最小基因集的信息，以及探索潜在的基因(或其他特征)邻接规则和其他基因组结构要求。该项目将首次允许人们提出关于染色体的基本属性、基因组组织、基因内容、RNA剪接功能、原核生物和真核生物的区别以及许多其他与进化有关的问题的深入问题。事实上，完全合成的染色体(最终是完全合成的基因组)的可获得性允许对进化问题进行直接测试，而这是任何其他方式都无法解决的。最终将被设计和提纯的“合成酵母”很可能会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卓越生物，具有广泛的实际用途，包括从农产品和副产品中生产乙醇。约翰·霍普金斯大学内外的无数教育机会都将从该项目中产生。除了新的课程内容和新的整体课程外，还将在发展中国家(印度)创建课程内容，并将启动有才华的当地高中生、高中教师和马里兰科学中心参与的活动，他们对该项目非常感兴趣。这门新课程的重点是让本科生直接参与一个大规模的功能基因组项目。

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.