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

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

Profiling Synaptic Proteins Identifies Regulators of Insulin Secretion and Lifespan

Cells are organized into distinct compartments to perform specific tasks with spatial precision. In neurons, presynaptic specializations are biochemically complex subcellular structures dedicated to neurotransmitter secretion. Activity-dependent changes in the abundance of presynaptic proteins are thought to endow synapses with different functional states; however, relatively little is known about the rules that govern changes in the composition of presynaptic terminals. We describe a genetic strategy to systematically analyze protein localization at Caenorhabditis elegans presynaptic specializations. Nine presynaptic proteins were GFP-tagged, allowing visualization of multiple presynaptic structures. Changes in the distribution and abundance of these proteins were quantified in 25 mutants that alter different aspects of neurotransmission. Global analysis of these data identified novel relationships between particular presynaptic components and provides a new method to compare gene functions by identifying shared protein localization phenotypes. Using this strategy, we identified several genes that regulate secretion of insulin-like growth factors (IGFs) and influence lifespan in a manner dependent on insulin/IGF signaling

Analysis of mRNA Abundance in <i>mec-3(e1338)</i> Animals

<p>Fold-change (<i>x</i>-axis) is plotted against the statistical significance (<i>y</i>-axis) for each probeset. Fold-changes are shown on log₂ scale. <i>p-</i>Values are shown on a negative log₁₀ scale. The symbol × indicates genes with reduced expression in <i>mec-3(e1338)</i> animals (fold-change < −1, <i>p</i> < 0.01). Light blue circles indicate genes with reduced expression that are also on Chromosome 4. Dark blue circles indicate genes with reduced expression that are within 1 cM to the left or right of <i>mec-3</i>. The open red circle indicates the <i>mec-3</i> gene.</p

Analysis of mRNA Abundance in the KP3365 <i>unc-43(n1186)</i> CaMKII Strain

<p>Expression data are illustrated as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0010002#pgen-0010002-g002" target="_blank">Figure 2</a>. The symbol × indicates probesets with reduced expression in KP3365 animals (fold-change < −0.5, <i>p</i> < 0.01). Filled blue circles indicate probesets with reduced expression that are also on Chromosome 4. Open red circles indicate probesets corresponding to the <i>unc-43</i> CaMKII gene. The black square indicates the probeset corresponding to the <i>hif-1</i> gene<i>.</i></p

Fraction of <i>C. elegans</i> Alleles That Are Nonsense Mutations

<p>Molecular information about alleles was obtained from WormBase and literature searches. Graph includes 117 genes for which molecular characterization of three or more alleles was available (770 alleles total). Of these 117 alleles, 22 (19%) have no known nonsense mutations. Many of these no-nonsense alleles are in genes that are required for viability.</p

Positional Cloning of <i>tom-1(nu468)</i>

<p>Expression data are illustrated as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0010002#pgen-0010002-g002" target="_blank">Figure 2</a>. The symbol × indicates probesets with reduced expression in KP3293 <i>nu468</i> (fold-change < −0.5, <i>p</i> < 0.01). Filled blue circles indicate probesets with reduced expression in KP3293 <i>nu468</i> that are also on Chromosome 1. The open red circle indicates the probeset corresponding to <i>tom-1.</i> Sequencing of the <i>tom-1</i> gene in KP3293 <i>nu468</i> revealed a W212Stop mutation in the <i>tom-1</i> gene (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0010002#pgen-0010002-g007" target="_blank">Figure 7</a>A).</p

Expression of TOM-1, the <i>C. elegans</i> Ortholog of Tomosyn

<p>(A) Schematic of worm tomosyn indicating the location of the premature stop found in <i>nu468</i> and deletion in <i>ok285</i>.</p> <p>(B–D) Expression pattern of <i>tom-1</i> characterized with 4.2 kb of sequence upstream of the start codon driving expression of green fluorescent protein. Expression is seen in ventral cord motor neurons, with cell bodies indicated by arrowheads (B) and a number of neurons in the head (C) and the tail (D). Scale bars = 10 μm.</p

Characterization of the Splicing Defect in <i>hif-1(nu469)</i>

<p>(A) Diagram of <i>hif-1</i> gene structure and the two mutations in <i>hif-1(nu469),</i> a previously uncharacterized lesion in the background of the KP3365 strain. This lesion consists of two closely linked mutations: (1) C→A at position 2315 of the coding sequence, resulting in a P771Q mutation, and (2) an insertion of TTATCA after position 2373. Sequencing cDNA from KP3365 revealed that these two mutations result in the inappropriate splicing of the <i>hif-1</i> transcript (indicated by a dashed line), removing 135 base pairs of the last exon. Location of primers for PCR are indicated by half-arrows above exons 9 and 11.</p> <p>(B) RT-PCR confirmed the altered splicing of the <i>hif-1(nu469)</i> mRNA.</p