Analysis of Gene Function by Parallel Phenotyping using Barcoded Mutants

拟议的研究计划是旨在了解分子的长期计划的一部分。 控制网柄苔藓发育的机制。我们的目标是突变基因并研究由此产生的 表型作为发现这些基因功能的途径。通过使用随机的组合 突变和定向敲除策略我们将在10,500个蛋白质中的大约一半产生突变 编码基因，其中每个突变用唯一的60个核苷酸的DNA序列(分子 条形码)。利用限制性内切酶诱导单倍体菌株(AX4)的插入突变 随机选择重组质粒DNA进行整合(REMI)，用质粒挽救法克隆。DNA序列 将确定每个克隆的侧翼，从而确定每个突变的插入位置，并将基因组 插图的位置将公布在项目网站(dictygenome.org)上，以便分发 突变体对基因敲除质粒进行蚂蚁攻击。我们还将使用我们开发的基于聚合酶链式反应的方法 敲除选定的一组基因，如编码蛋白激酶、转录因子和 推测为细胞黏附和识别受体。作为基因功能的一种衡量标准，我们将确定 每个突变体在768混合体中执行各种发育和生长阶段功能的能力 变种人。在这些竞争性的表型实验中，每个突变体都将通过其 条形码DNA标签到条形码寡核苷酸微阵列，在PCR扩增所有条形码后 混合物。例如，一套完整的突变体将经历几个生长周期， 发育、产孢子和萌发，DNA样本将从每个存活的突变株中提取 连续的一步。从这个群体中退出的突变体，但仍然存在于控制的细胞群体中 在没有干预发育周期的情况下繁殖的将被记录为发育缺陷。 将开发细分为可定义步骤的附加实验(聚合、段塞迁移等) 将进一步缩小突变表型的范围。我们将使用这些并行执行额外的功能测试 分析方法，并在适当的情况下，通过对单个突变进行表型鉴定。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 Iii)可以通过未来的实验进行测试。

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.