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note
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b or pAct alone; 0.01 μ,g pAct-tim and pAct-per or 0.02 μg pAct; 0.0005 μg pAct-clock; and 0.5 μg pAct. Cells were kept at 22°C in continuous light (40 μmol). The dark-cultured samples were wrapped in foil and kept in the same chamber. Cells were harvested 48 hours after transfection, and enzyme activity was measured with Dual-Luciferase Reporter Assay (Promega), β-Galactosidase Enzyme Assay System (Promega), and Galacto-Light Plus (Tropix) as appropriate. For each sample, reporter activity was normalized to control enzyme activity. Reporter activity is plotted relative to activity when co-transfected with pAct-clock. Values are the mean ± SEM of 4 to 6 replicate experiments.
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31
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0344364393
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A stable S2 cell line expressing full-length tim under the Drosophila actin 5C promoter was generated essentially as described (30) with Lipofectin (GIBCO-BRL) as the transfection reagent and 20 to 1 ratio pAct-tim to pCopHyg. This TIM stable cell line was then transiently transfected with Effectene (Qiagen) with 0.4 μg of pAct-cry-gfp alone or together with 0.4 μg of pAct-per (pAct was used to keep the amount of DNA per well constant). Free GFP expressed under the same promoter served as negative control. Cells were kept at 22°C under constant light condition (∼50 μmol); the dark control plates were wrapped in foil during the whole experiment, and cells were harvested under red safe light; the 1-hour light pulse was performed under the same conditions and cells were harvested immediately after.
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32
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0344364392
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In order to detect CRY, we tagged it with GFP, generating a CRY-GFP fusion (32). When used in place of pAct-cry in experiments such as those of Fig. 1, pAct-cry-gfp generated indistinguishable results, thus arguing that the fusion protein can adopt a functional conformation (21).
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33
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0345659003
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6 cells for each immunoprecipitation reaction. ColP assays were performed as in (33) whenever Gammabind (Pharmacia) was used, or following the manufacturer's recommendation in the experiments including anti-ratsepharose (Sigma). After 48 hours, transfected cells were washed twice in PBS and lysed in 20 mM Hepes pH 7.5; 100 mM KCl; 0.05% Triton X-100; 2.5 mM EDTA; 5 mM DTT; 5% glycerol; aprotinin (10 μg/ml); leupeptin (10 μg/ml); and pepstatin (2 μg/ml). Each colP experiment was repeated four times. Proteins were loaded into a precast 4 to 15% acrylamide gel (BioRad). Protein immunoblots were performed with 1/2000 dilution of rat antibody to TIM (16) or rabbit antibody to GFP (Molecular Probes), followed by HRP-conjugated secondary antibodies. The signal was visualized with the ECL kit (Amersham).
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34
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0344796221
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2] separating the LEXA and CRY sequences; a direct amino-terminal fusion of LEXA to CRY showed the same results but produced distinctly weaker signals. Yeast were cotransformed with three selectable expression plasmids, a LEXA plasmid (TRP1 marker), a VP16 plasmid (LEU2 marker), and pMET25::ADE2 (ADE2 marker), either nonrecombinant or containing a full-length tim cDNA. pMET25::ADE2 was derived from p426MET25 (34) by removing the Ndel-Kpal URA3 fragment and replacing it with a 3.6-kb Bam HI ADE2 fragment by blunt-ligation. For light and dark growth conditions, each yeast transformation reaction was split onto two plates of selection medium. The plates were incubated next to one another at 30°C under a NEC B-W Cool White fluorescent light source (38-cm distance), but one was wrapped in foil to block the light. After 3 days, yeast colonies from each condition were patched in duplicate onto two fresh selection plates and grown for 1 to 2 days in light or dark as above. From each duplicate pair of plates from each condition, one was used for two-hybrid interaction assays and one for immunoblot analysis. For immunoblot analysis, light-and dark-grown patches were inoculated into 2 ml of liquid selection medium and shaken overnight in light or dark as above. Cultures were diluted into 20 ml of selection medium, grown in light or dark as above to an optical density of ∼0.7, and total protein was determined (Bio-Rad). 3-ml aliquots were pelleted, dissolved in 70 μl of Laemmli buffer, and diluted in Laemmli buffer to equalize the total protein concentration of the different samples. Samples were run on 7.5% SDS-PAGE, blotted onto nitrocellulose, and immunodetection performed with rabbit anti-LEXA antiserum (1:2000) and goat anti-rabbit IgG-HRP (1:5000). Signals were detected by enhanced chemiluminesence (Amersham).
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35
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0345659002
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unpublished observations
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D. Staknis and C. Weitz, unpublished observations.
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Staknis, D.1
Weitz, C.2
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36
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0344796220
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In additional experiments of slightly different design, no interaction was detected between LEXA-PER (full length) and VP16-CRY under conditions in which interactions were detected between LEXA-PER (full length) and VP16-TIM and between LEXA-TIM and VP16-CRY.
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0345659001
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Cells in a 12-well plate setting were transfected as above (22) and kept under constant conditions covered in foil until the end of the experiment. In a blind experiment 100 cells corresponding to each treatment were analyzed. For nuclear staining, cells were embedded in a solution containing 100 nM DAPI (Molecular Probes) and 200 nM propidium iodide (Molecular Probes). Confocal and conventional fluorescence microscopy were performed in an Olympus IX70 inverted microscope equipped with a 100 X objective. Fluorescence was filtered with FITC filter sets (Olympus).
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41
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0344364391
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We engineered Xho I and Hind III sites at the 5' and 3' ends of the CRY ORF in HL02979 (Berkeley Drosophila Genome Project/HHMI EST Project, unpublished data), respectively. The resulting cry-gfp fusion was subcloned as a blunt fragment into pAct (19).
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We thank K. Wager-Smith, A. Schinder, J. Hogenesch, D. Somers for critical reading of the manuscript and S. Harmer for helpful discussions; J. Sheen for the CFP clone (blue-sGFP-TYG-nosKS) (35), J. Blau, L. Saez, and M. Young for the TIM antibodies; R. Brent for the anti-LEXA antiserum; and J. Mosher and S. Crews for pRL-copia. This work was supported by National Institute of Mental Health grant MH-51573 and the NSF Center for Biological Timing (both to S.A.K.), Pew Foundation (as a fellowship to M.F.C.), and National Institute of Mental Health grant MH-59943 and the Harvard-Armenise Foundation for Advanced Scientific Research (both to C.J.W.).
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