Analysisof Gene Function by Parallel Phenotyping using Barcoded Mutants

Baylor College Of Medicine, Houston TX

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Abstract

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