Using Microarrays to Facilitate Positional Cloning: Identification of Tomosyn as an Inhibitor of Neurosecretion

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. Genetic screens are commonly used to figure out which genes are involved in a biological process. The first step in a genetic screen is to isolate mutant animals that are defective in the process being studied. The next step is to find which of the thousands of genes has the mutation that causes the observed defect. Positional cloning, the tried-and-true method for locating mutations, is slow and expensive. The authors propose using microarray hybridizations to speed the process. Their approach relies on the fact that a large fraction of the mutations found in screens are the results of premature stop codons, a particularly severe type of mutation. In cells, messages containing premature stop codons are rapidly destroyed by a protective pathway, called nonsense-mediated decay, thus making them directly detectable by microarray hybridization. The authors apply this strategy retrospectively to known mutants in Caenorhabditis elegans, Arabidopsis, and mouse. They identify two uncharacterized mutations in C. elegans, including one, tom-1, found in a forward genetic screen for enhancers of neurotransmission. Interestingly, their characterization of tom-1 mutants suggests that the highly conserved protein tomosyn inhibits neurotransmission in neurons. This study shows that microarray hybridizations will help reduce the time and effort required for positional cloning.