GENERATION OF THE COMPLETE SET OF YEAST GENE DISRUPTIONS

描述：(申请人摘要)最近确定的 面包酵母(S.cerevisiae)的基因组显示，几乎三分之二的 这种经过充分研究的有机体的基因没有被发现。因此，我们 对构成的大多数基因的功能几乎一无所知 这个简单的真核细胞。催化接近最终目标 全面了解所有蛋白质的功能 构建真核细胞，我们建议快速构建完整的 大约6000个基因中的每一个都被删除的一组突变 重要的模式生物。似乎可以肯定的是，这种广泛的分布 突变株的收集将大大加快基因发现的步伐 功能。 这一目标将由一个由5个实验室组成的联盟来实现，每个实验室 在两年中每年扰乱300个基因，产生3000个突变。另一个 生物体的3000个基因将被我们的欧洲人和加拿大人破坏 同事们。将对突变体进行几种标准表型的筛选，以 为突变集合的用户提供必要的信息，然后 广泛分发给所有感兴趣的科学家，以获得更详细和 完整的分析。 我们建议用KanMx基因替换每个基因的编码序列 编码对庆大霉素的耐药性。此外，一种新的序列 标签将被引入到每个突变体中，以唯一地识别它们。 在两种交配类型的单倍体中，非必需基因将被破坏； 必需和近乎必需的基因将在二倍体中被破坏。基因 颠覆将使用简单和流行的基于聚合酶链式反应的 这项技术将使构建完整的突变体集成为可能 两年了。在项目的最后一年，我们将提升 通过构造有条件表达的 必需和近乎必需的基因，构建了大约750个双倍 重复基因对的两个成员的突变都被删除，并扰乱了 小基因，因为它们被揭示。

DESCRIPTION: (Applicant's abstract) The recently-determined sequence of the genome of bakers' yeast (S. cerevisiae) revealed that almost two-thirds of the genes of this well-studied organism had escaped detection. Thus, we know almost nothing about the function of most of the genes that comprise this simple eukaryotic cell. To catalyze approaching the ultimate goal of a comprehensive understanding of the function of all the proteins that constitute a eukaryotic cell, we propose to construct quickly the complete set of mutants deleted for each of the approximately 6000 genes of this important model organism. It seems certain that wide distribution of this collection of mutants will greatly accelerate the pace of discovery of gene function. This goal will be achieved by a consortium of 5 laboratories, each disrupting 300 genes in each of two year, yielding 3000 mutants. The other 3000 genes of the organism will be disrupted by our European and Canadian colleagues. The mutants will be screened for a few standard phenotypes to provide necessary information for the users of the mutant collection, then distributed widely to all interested scientists for more detailed and complete analysis. We propose to replace the coding sequence of each gene with the KanMx gene encoding resistance to the drug gentamycin. In addition, a novel sequence tag will be introduced into each of the mutants to uniquely identify them. Non-essential genes will be disrupted in haploids of both mating types; essential and nearly essential genes will be disrupted in diploids. Gene disruption will be accomplished using a facile and popular PCR-based technique that will enable construction of the complete set of mutants in two years. In the final year of the project, we will enhance the value of the mutant collection by constructing conditionally-expressed versions of essential and nearly essential genes, constructing approximately 750 double mutants deleted for both members of a duplicated gene pair, and disrupting small genes as they are revealed.