The third mutation does not shake under ether anesthesia and complements Shaker. This mutation, insomniac (inc), causes a severe reduction of sleep to an average of 317 min per day, over four standard deviations from the mean of all screened lines ( Figure 1A) and a > 65% reduction from that of wild-type CS control animals, which average 927 min of sleep per day ( Figure 1B). Individual mutant animals exhibit strikingly reduced sleep during both day and night ( Figure 1C), but do not display other obvious behavioral (including geotaxis and

phototaxis) or morphological abnormalities. We mapped insomniac to a region of 250 kb–1 Mb near the tip of the X chromosome (see Figure S1A available online and Experimental Procedures), and further analysis identified a deficiency, IDO inhibitor removing 190 kb and nine annotated genes, that failed to complement insomniac ( Figure S1B). Coding regions of these candidate genes, as well as some introns and intergenic regions, were amplified from insomniac and CS animals and buy GSK1349572 sequenced. A single mutation was identified, a 257 bp deletion between two divergently transcribed genes, CG14795 and CG32810 ( Figures 2A–2C). This deletion is not present in other wild-type strains, and complete sequencing of CG32810 and CG14795 revealed no other alterations in either gene (data

not shown). To map the 5′ termini of CG32810 and CG14795 with respect to the deletion, we performed 5′ RACE. This analysis indicated that the deletion removes the transcriptional initiation site of CG32810 and 50 bp of its 5′UTR, but leaves intact the 5′ terminus of CG14795 and a small amount of upstream sequence ( Figure 2B). In insomniac animals, discrete 5′ RACE products were reduced however or absent for both CG32810 and CG14795, indicating that the transcription of both genes is affected by the deletion ( Figure 2D). To further assess whether disruption of one or both genes causes the insomniac phenotype, we obtained a transposon insertion located in the 5′UTR

of CG32810 (CG32810f00285; Figures 2A and 2B). As described below, molecular and behavioral analysis of these mutations indicates that CG32810 corresponds to insomniac, and we therefore refer to the 257 bp deletion as inc1 and the transposon insertion as inc2. To quantitatively compare the effects of these mutant alleles on transcript levels of CG32810 and CG14795, we prepared RNA from whole animals, as well as from isolated heads and bodies, and performed northern blotting analysis using probes specific for each transcript and for rp49, a control gene. The inc1 deletion is associated with a severe (>90%) decrease in CG32810 transcript levels and a substantial (∼60%) reduction in those of CG14795 ( Figure 2E and data not shown), consistent with the reduced transcript levels observed in 5′ RACE analysis ( Figure 2D).