Analysis of Gene Function by Parallel Phenotyping using Barcoded Mutants

拟议的研究计划是一项长期计划的一部分，旨在了解分子生物学。 控制网骨藻发育的机制我们的目标是使基因突变， 表型作为发现这些基因功能的途径。通过使用随机的 通过诱变和定向敲除策略，我们将在10，500个蛋白质中的约一半中产生突变， 编码基因，其中每个突变都用独特的60个核苷酸的DNA序列（分子标记）标记 条形码）。插入突变将通过限制性内切酶介导在单倍体菌株（AX4）中诱导， 随机选择质粒DNA，并通过质粒拯救克隆。的DNA序列 将确定每个克隆的侧翼，并因此确定每个突变的插入位点， 插入的位置将公布在项目网站（dictygenome.org）上， 突变体和敲除质粒。我们还将使用我们开发的基于PCR的方法， 敲除选定的基因组，例如编码蛋白激酶、转录因子和 假定的细胞粘附和识别受体。作为基因功能的一种衡量标准，我们将确定 每种突变体在768种混合物中执行各种发育和生长阶段功能的能力 变种人在这些竞争性表型分析实验中，每个突变体将通过其 在PCR扩增条形码寡核苷酸微阵列中的所有条形码之后， 混合物.例如，一套完整的突变体将经历几个生长周期， 发育，孢子形成和萌发，DNA样品将从每个存活的突变体中制备 连续步骤。突变体从这个群体中退出，但在对照细胞群体中持续存在， 在没有发育周期干预的情况下繁殖，将被记录为发育缺陷。 将开发细分为可定义步骤（聚合、段塞运移等）的附加实验 将进一步缩小突变体的表型我们将使用这些并行的 分析方法，并在适当的情况下，通过对单个突变体进行表型分析。将这些结果与 转录表型分析（项目II）的结果将使我们能够提出调控网络（项目II）。 （3）可以通过未来的实验进行验证。

The proposed research plan is part of a long-term program aimed at understanding the molecular mechanisms that control development in Dictyostelium. We aim to mutate genes and study the resulting phenotypes as an avenue to discovering the function these genes. By using a combination of random mutagenesis and directed knockout strategies we will generate mutations in about half of the 10,500 protein coding genes where each mutation is tagged with a unique 60-nucleotide DNA sequence (a molecular barcode). Insertion mutations will be induced in a haploid strain (AX4) by restriction enzyme mediated ntegration (REMI) of plasmid DNA, selected at random, and cloned by plasmid rescue. The DNA sequence flanking each clone, and therefore the insertion site of each mutation, will be determined and the genomic ocations of the insertions will be published to the project website (dictygenome.org) for distribution of the mutants ant the knockout plasmids. We will also use a PCR-based method that we have developed to knockout selected cohorts of genes, such as those encoding protein kinases, transcription factors and putative cell adhesion and recognition receptors. As one measure of gene function, we will determine the ability of each mutant to carryout various developmental and growth-stage functions in mixtures of 768 mutants. In these competitive phenotyping experiments, each mutant will be detected by hybridization of its barcode DNA tag to a barcode oligonucleotide microarray, after PCR amplification of all barcodes in the mixture. For example, a complete set of mutants will be taken through several cycles of growth, development, sporulation and germination, and DNA samples will be made from the mutants surviving each successive step. Mutants that drop out of this population, but persist in a control population of cells that were propagated without intervening cycles of development will be recorded as developmentally defective. Additional experiments that sub-divide development into definable steps (aggregation, slug migration, etc.) will further narrow the mutant phenotypes. We will carryout additional functional tests using these parallel analysis methods and, when appropriate, by phenotyping individual mutants. Integrating these results with the results of the transcriptional phenotyping (Project II) will allow us to propose regulatory networks (Project III) that can be tested by future experiments.