Aldicarb is a reversible acetylcholinesterase inhibitor that increases the accumulation of acetylcholine in the synaptic cleft causing whole body paralysis and inhibition of pharyngeal pumping. Homozygous gei-8(ok1671) mutants (n=64) and wild-type animals (n=75) at the L4 stage were incubated on NGM plates with 1 mM aldicarb and scored over selleck chemical time for paralysis in three separate experiments. The onset of paralysis occurred significantly earlier in gei-8(ok1671) mutants than in wild-type controls (Figure 7A). Levamisole is a cholinergic agonist that also results in animal paralysis. We performed two experiments with homozygous gei-8(ok1671) mutants (n=40) and wild-type animals (n=40) at the L4 stage on NGM plates with levamisole at a concentration of 1 mM.

As in the aldicarb assay, the onset of paralysis occurred significantly earlier in gei-8(ok1671) mutants compared to controls (Figure 7B). Taken together, these results indicate that the gei-8(ok1671) mutation results in abnormal cholinergic signaling, however, it does not distinguish between post-synaptic versus pre-synaptic transmission defects. Figure 7 Analysis of neuromuscular function of gei-8(ok1671) mutant (VC1213). gei-8 Loss of Function Leads to Transcription Deregulation Effects of the gei-8(ok1671) mutation on gene expression were studied with whole genome microarrays (Affymetrix). Changes in gene expression were defined as increased or decreased if statistically significant compared to wild-type controls in at least 2 out of 3 biological replicates.

Deregulated genes were analyzed for Gene Ontology (GO) term enrichment and clustered according to functional classification using DAVID 6.7 [40] and KEGG pathway tools [41]. Expression microarray analysis revealed 756 probe sets with decreased expression, corresponding with 690 unique Wormbase IDs (Table S1). DAVID classification tools [40] identified 645 IDs using medium classification stringency. GO analysis resulted in 32 clusters with an enrichment score greater than 2 and P<0.05. The list was enriched in spliceosome (29 genes), proteasome (13 genes), cysteine and methionine metabolism (7 genes), and RNA polymerase genes (6 genes) as identified by KEGG pathway analysis. Among specific genes involved are RNA polymerase II and III (Pol II subunits B4, B7, B9 and Pol III subunits AC2 and F09F7.

3), spliceosome components (U1 to U6 snRNAs, hel-1 helicase and others), and proteasome subunits (pas-3, pas-4, pbs-1, pbs-3, pbs-4, pbs-6, Batimastat pbs-7, rpt-1, rpt-2, rpn-2, rpn-5, rpn-8, rpn-12). The most common functional categories over represented by the changes in gene expression were growth, embryonic or larval development and development of reproductive structures. Other clusters include multiple histones and histone-like genes, mitochondrial membrane proteins, sperm structural proteins and hedgehog-like family genes.