Abstract
Forward genetic screens have been used as a powerful strategy to dissect complex biological pathways in many model systems. A significant limitation of this approach has been the time-consuming and costly process of positional cloning and molecular characterization of the mutations isolated in these screens. Here, the authors describe a strategy using microarray hybridizations to facilitate positional cloning. This method relies on the fact that premature stop codons (i.e., nonsense mutations) constitute a frequent class of mutations isolated in screens and that nonsense mutant messenger RNAs are efficiently degraded by the conserved nonsense-mediated decay pathway. They validate this strategy by identifying two previously uncharacterized mutations: (1) tom-1, a mutation found in a forward genetic screen for enhanced acetylcholine secretion in Caenorhabditis elegans, and (2) an apparently spontaneous mutation in the hif-1 transcription factor gene. They further demonstrate the broad applicability of this strategy using other known mutants in C. elegans, Arabidopsis, and mouse. Characterization of tom-1 mutants suggests that TOM-1, the C. elegans ortholog of mammalian tomosyn, functions as an endogenous inhibitor of neurotransmitter secretion. These results also suggest that microarray hybridizations have the potential to significantly reduce the time and effort required for positional cloning.
Highlights
Forward genetic screens have been traditionally utilized in model systems (e.g., Caenorhabditis elegans, Drosophila, yeast, and Arabidopsis)
(1) How frequently are nonsense alleles recovered in forward genetic screens? (2) Are microarray hybridizations sensitive enough to detect the decreased abundance of a nonsense mutant transcript? (3) Can microarray hybridizations be used to identify an uncloned behavioral mutant in C. elegans? (4) Is this microarray-based strategy applicable to other model organisms?
To assess the prevalence of nonsense alleles isolated following random mutagenesis, we compiled a list of sequenced C. elegans mutant alleles by downloading information from WormBase and conducting targeted literature searches (Figure 1; Table S1)
Summary
Forward genetic screens have been traditionally utilized in model systems (e.g., Caenorhabditis elegans, Drosophila, yeast, and Arabidopsis). Mutations isolated in genetic screens are typically identified by positional cloning. The ultimate goal of a typical positional cloning project is to analyze a sufficient number of recombinants to map the mutation to a small genetic interval (typically approximately 0.1 cM). The difficulty of a particular positional cloning can be compounded by the nature of the mutant phenotype. This problem is acute for behavioral mutants, which often have phenotypes that must be scored in multiple trials, or in populations of animals. Together, these issues conspire to make traditional positional cloning a significant and costly bottleneck
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