Gene discovery by chemical mutagenesis and whole-genome sequencing in Dictyostelium.
Whole-genome sequencing is a useful approach for identification of chemical-induced lesions, but previous applications involved tedious genetic mapping to pinpoint the causative mutations. We propose that saturation mutagenesis under low mutagenic loads, followed by whole-genome sequencing, should allow direct implication of genes by identifying multiple independent alleles of each relevant gene. We tested the hypothesis by performing three genetic screens with chemical mutagenesis in the social soil amoeba Dictyostelium discoideum Through genome sequencing, we successfully identified mutant genes with multiple alleles in near-saturation screens, including resistance to intense illumination and strong suppressors of defects in an allorecognition pathway. We tested the causality of the mutations by comparison to published data and by direct complementation tests, finding both dominant and recessive causative mutations. Therefore, our strategy provides a cost- and time-efficient approach to gene discovery by integrating chemical mutagenesis and whole-genome sequencing. The method should be applicable to many microbial systems, and it is expected to revolutionize the field of functional genomics in Dictyostelium by greatly expanding the mutation spectrum relative to other common mutagenesis methods.
Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas 77030, USA;
Cheng-Lin Frank
CL
Li
Graduate Program in Structural Computational Biology and Molecular Biophysics, Baylor College of Medicine, Houston, Texas 77030, USA;
Balaji
B
Santhanam
Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas 77030, USA;
Amanda Nicole
AN
Webb
Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas 77030, USA; Faculty of Computer and Information Science, University of Ljubljana, SI-1000 Ljubljana, Slovenia.
Blaž
B
Zupan
Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas 77030, USA;
Gad
0000-0002-0532-0551
G
Shaulsky
D002874:Chromosome Mapping; D004023:Dictyostelium / Q000187:drug effects / Q000235:genetics*; D056726:Genetic Association Studies; D059014:High-Throughput Nucleotide Sequencing; D016296:Mutagenesis / Q000187:drug effects / Q000235:genetics*; D009153:Mutagens / Q000633:toxicity; D000073336:Whole Genome Sequencing / Q000379:methods*
D009153:Mutagens