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.
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