Phase shifting of the circadian clock by induction of the Drosophila period protein

Science

Volumn 263, Issue 5144, 1994, Pages 237-240

  Edery, Isaac ^a,b   Rutila, Joan E ^b   Rosbash, Michael ^b  

^a RUTGERS UNIVERSITY   (United States)

^b BRANDEIS UNIVERSITY   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

ARTICLE; CIRCADIAN RHYTHM; DROSOPHILA; NONHUMAN; PRIORITY JOURNAL; PROTEIN INDUCTION; TRANSGENIC ANIMAL;

EID: 0028158263     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.8284676     Document Type: Article

References (34)

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20. +;hspc-23a flies. Flies were first subjected to four cycles of 12 hours of light and 12 hours of dark followed by three continuous 24-hour dark periods. On the fourth dark period, flies were incubated for 1 hour at 37°C, and subsequent behavioral data were collected for each fly (25). Only data from those flies that fulfilled the following criteria were used for Fig. 2: (i) endogenous periods between 23.5 and 24.5 hours; (ii) no changes in endogenous periods greater than ±0.5 hour after the temperature shift; and (iii) strong rhythms [that is, a power setting greater than 10 (25)] before and after the temperature shift. These criteria ensured that PRCs (Fig. 2) were derived from flies with significant locomotor activity rhythms that also did not experience stable period changes after the temperature pulse. The circadian time is almost identical to Zeitgeber time, and we therefore use the term circadian time in this context. We determined the activity phase before and after the temperature pulse by identifying the activity offset for each activity peak, after the data had been filtered as described (25). The activity offset was defined as the time where the activity was 50% of the peak value, after that peak had occurred. The location where the next "average" phase offset would occur, had a temperature shift not been imposed, was then calculated by the method of least squares. The activity profile for the day when the temperature pulse was applied was not included in this average phase determination. The difference between the average phase offset before and after the heat pulse is the phase shift.
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23. +;hspc-23a flies to conditions identical to those of flies used in our behavioral studies (Fig. 2). At the appropriate times (Fig. 3), flies were frozen and heads collected. Approximately 30 to 50 heads were sonicated in 200 μl of buffer I [100 mM KCl, 20 mM Hepes (pH 7.5), 5% glycerol, 10 mM EDTA, 1% Triton X-100, 0.1% SDS, 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride (PMSF), aprotinin (20 μg/ml), leupeptin (2 μg/ml), and pepstatin A (1 μg/ml)]. Equal amounts of supernatant protein were mixed with SDS sample buffer, boiled, and analyzed by protein immunoblot (77).
24. All gels were 6% (29.6% acrylamide and 0.4% bis-acrylamide) polyacrylamide-SDS and cast in a Bio-Rad mini-PROTEAN II apparatus. After electrophoresis, the gels were electroblotted onto nitrocellulose for 23 min at 0.24 A with a semidry blotting apparatus according to the manufacturer's specifications (Bio-Rad). After the blot was washed in TBST [10 mM tris-HCl (pH 7.5), 140 mM NaCl, and 0.1% Tween-20], it was incubated for 1 hour in TBST containing 1% bovine serum albumin (BSA). An antibody to PER (78) was added at a dilution of 1:2000 in 1% BSA (TBST) for 3 to 5 hours. Extensive washing in TBST was followed by incubation with a 1:3000 dilution of an antibody to rat immunoglobulin G conjugated to horseradish peroxidase (Amersham) for 20 min in 1% BSA (TBST). After another extensive wash in TBST, the blots were visualized by chemiluminescence (Amersham).
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34. We thank J. C. Hall for many helpful comments on the manuscript, R. Peterson and S. Lindquist for the plasmid p70 ATG, D. A. Wheeler for help with the FORTRAN programming, H. V. Colot for proofreading, and L. A. Monaghan for secretarial assistance.