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Science 18 July 2008:
Vol. 321. no. 5887, pp. 372 - 376
DOI: 10.1126/science.1155942
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Research Articles

Identification of SLEEPLESS, a Sleep-Promoting Factor

Kyunghee Koh1*, William J. Joiner1*, Mark N. Wu2*, Zhifeng Yue1, Corinne J. Smith1 and Amita Sehgal1{dagger}

1 Howard Hughes Medical Institute, Department of Neuroscience, University of Pennsylvania, Philadelphia, PA 19104, USA.
2 Division of Sleep Medicine, Department of Neurology, University of Pennsylvania, Philadelphia, PA 19104, USA.


Figure 1 Fig. 1. Sleep phenotype of sss mutants. (A) Histogram showing the distribution of daily sleep for ~3500 mutant lines (~8 female flies per line). For each line, daily sleep is shown as the difference from the mean of a group of about 100 to 250 lines tested simultaneously. The arrow indicates the sss mutant line. (B) Sleep profile in 30-min intervals for sss flies (open diamonds) versus background controls (ctrl, solid diamonds). Data for male (M) and female (F) flies are shown. The bar below the x axis indicates 12-hour light and 12-hour dark periods. (C) Daily sleep amount for control (162 males and 148 females), control/sss (111 males and 113 females), and sss flies (146 males and 148 females). Data from the same flies are shown in (C) to (F). (D to F) Activity counts per minute awake (D), sleep bout duration (E), and daily number of sleep bouts (F) for male and female control, control/sss, and sss flies. In this and subsequent figures, error bars represent SEM. *P <0.05, **P < 0.0001. For (C), (E), and (F), significance level is shown for sss mutants compared to both control and control/sss flies. For (D), significance level is shown for pairwise comparisons as indicated by lines. In (E), sleep bout duration, which is not normally distributed, is presented as simplified box plots. The line inside each box indicates the median; the top and bottom represent 75th and 25th percentiles, respectively. About 9% of flies exhibiting zero sleep were excluded from calculation of sleep bout duration. [View Larger Version of this Image (30K GIF file)]
 

Figure 2 Fig. 2. sss encodes a brain-enriched, GPI-anchored protein. (A) Schematic of the genomic structure of the sss locus. Noncoding regions of the cDNA are shaded; coding regions are shown in white. (B) Schematic of structural features of the SSS protein. The primary sequence contains a predicted signal peptide, an N-type glycosylation site ({psi}), and a potential GPI attachment site (*). (C) Amino acid sequence of SSS (GenBank accession number EU816195). Amino acids 1 to 32 constitute the predicted signal peptide (boxed); the predicted N-type glycosylation site is underlined. Asterisk denotes the predicted GPI attachment site. Abbreviations for amino acids: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; Y, Tyr. (D) Glycosylation of the SSS protein. Western blot analysis with SSS antibody revealed two bands detected in head extracts from wild-type (ctrl) but not sss flies. Deglycosylation of head extracts by treatment with PNGase F resulted in detection of a single band. Because our antibody to SSS does not recognize glycosylated SSS well, Western blots were treated with peptide N-glycosidase F (PNGase F) before being probed with antibody to SSS. In this and subsequent Western blots, antibody to mitogen-activated protein kinase (MAPK) was used to control for loading. (E) Surface expression of SSS in cultured Drosophila cells. S2R+ cells were transfected with a pIZ-sss construct and stained with or without permeabilization to assay for total or surface expression, respectively. Transfection with the pIZ vector alone shows specificity of our SSS antibody. (F) Reduced surface expression of SSS after PI-PLC treatment. S2R+ cells transfected with a pIZ-sss construct were stained without permeabilization after PI-PLC (+) or mock (–) treatment. (G) Release of SSS into the culture medium by PI-PLC. Western blot analysis of S2R+ cells transfected with pIZ-sss was performed after PI-PLC (+) or mock (–) treatment. (H) Enrichment of SSS expression in brain and head versus body. An equal amount of total protein (~40 µg) was loaded per lane. The experiments in (D) through (H) were performed three or four times with similar results. [View Larger Version of this Image (46K GIF file)]
 

Figure 3 Fig. 3. Genetic analysis of sss. (A) Daily sleep amount for precise excision (Pr, n = 26), sss{Delta}40 imprecise excision (Im, n = 15), precise/sssP1 (Pr/sssP1, n = 24), and imprecise/sssP1 (Im/sssP1, n = 35) female flies. (B) Western blot analysis of SSS protein levels. Similar levels of SSS protein are seen in head extracts from background control (ctrl) and precise excision (Pr) flies. SSS protein is undetectable in sssP1 and sss{Delta}40 imprecise excision (Im) flies. Similar results were obtained in two additional experiments. (C) Daily sleep amount for female sssP1 mutant flies with (TG1, n = 15; TG2, n = 8; TG3, n = 16) or without (n = 16) agenomic sss transgene. TG1, 2, and 3 refer to three independent transgene insertions, and one or two copies of the transgene were present in the flies tested. (D) Daily sleep amount for sssP2 (n = 110) versus background control (ctrl, n = 80) as well as control/sssP1 (n = 80) versus sssP2/sssP1 (n = 112) female flies. (E) Reduced levels of SSS protein in sssP2 and trans-heterozygous sssP2/sssP1 flies. Similar results were obtained in three additional experiments. Data from male flies of the genotypes shown in (A), (C), and (D) are shown in fig. S2. *P < 0.05, **P < 0.0001. [View Larger Version of this Image (13K GIF file)]
 

Figure 4 Fig. 4. Reduced homeostatic response to sleep deprivation in female sss mutants. (A) Amount of sleep lost during 6 or 12 hours of deprivation by the end of the dark period for background control (ctrl), sssP2, control/sssP1, and sssP2/sssP1 flies. Data from 13 to 56 female flies are shown. (B) Amount of sleep gained during 6 hours of recovery after deprivation as in (A). (C) Change in sleep latency after deprivation relative to undisturbed controls as in (A). Sleep latency is defined as the time between the end of deprivation (which coincided with light onset) and the start of a sleep bout. Data from male files are shown in fig. S3. *P <0.05, **P < 0.001. [View Larger Version of this Image (20K GIF file)]
 

Figure 5 Fig. 5. Circadian rhythm and longevity phenotypes of sss mutants. (A) Average activity records for background control (ctrl, n = 64) and sssP1 male flies (n = 81) assayed in constant darkness (DD). The activity records are double-plotted so that each horizontal line represents data for 2 days. The gray and black bars above each activity record indicate subjective day and night, respectively. (B) Activity records showing average activity in DD for control/sssP1 and control/sssP2 (n = 76) versus sssP2/sssP1 (n = 65) male flies. Circadian data for control/sssP1 and control/sssP2 flies were statistically similar and thus were pooled. (C) Cycling of PER protein in large ventral lateral neurons in control and sssP1 mutants. Ventral lateral neurons for control and sssP1 flies were stained for PER at indicated Zeitgeber times (ZT). PER protein levels are elevated at ZT2 and ZT20 and are low at ZT8 and ZT14. (D) Survivorship curves of background control (solid diamonds) and sssP1 (open diamonds) flies. Female sss flies (n = 187) show a significantly shorter life span (P < 0.0001) than controls (n = 198). Data from male flies are shown in fig. S5. [View Larger Version of this Image (59K GIF file)]
 

Figure 6 Fig. 6. sss is allelic to qvr and affects Sh expression. (A) Daily sleep amount for qvr (n = 31), versus background control (n = 32) as well as control/sssP1 (n = 30) versus qvr/sssP1 (n = 32) female flies. **P < 0.0001. (B) Altered sss transcripts in qvr mutants. RT-PCR products were obtained with qvr and background control (ctrl) RNA and water was used as a negative control (neg). (C) Schematic representation of sss transcripts in qvr mutants. qvr 1, 2, and 3 correspond to the top, middle, and bottom bands, respectively. In background control transcripts, 163 nucleotides of intron 6 are spliced out. In contrast, the entire intron is present in qvr 1 transcripts. In qvr 2 and 3 transcripts, splice donor sites differ from the one used in wild-type control transcripts, as indicated by the nucleotide numbers for splice sites. (D) Sequence change in qvr genomic DNA in intron 6 of sss. The fifth nucleotide in intron 6 has a G -> A transition. (E) Altered expression of SSS in qvr mutants. Fly head extracts from background control, qvr, and sssP1 flies were analyzed by Western blotting with SSS antibody. (F) Reduced expression of Sh in sss mutants. Western blot analysis of head extracts with Sh antibody reveals a Sh-specific band that is substantially reduced in sssP1 mutants relative to background control flies. Sh14 flies were used to identify a Sh-specific band, and Hk1 flies were used as an additional control. Nonspecific bands (*) may have obscured additional Sh bands. The experiments in (E) and (F) were performed three times with similar results. [View Larger Version of this Image (31K GIF file)]
 

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