Light-induced degradation of TIMELESS and entrainment of the drosophila circadian clock

Science

Volumn 271, Issue 5256, 1996, Pages 1736-1740

  Myers, Michael P ^a   Wager Smith, Karen ^a   Rothenfluh Hilfiker, Adrian ^a   Young, Michael W ^a  

^a ROCKEFELLER UNIVERSITY   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

NUCLEAR PROTEIN;

ANIMAL TISSUE; ARTICLE; AUTOREGULATION; BRAIN CELL; CIRCADIAN RHYTHM; CONTROLLED STUDY; DROSOPHILA; EYE; FEEDBACK SYSTEM; LIGHT; NONHUMAN; PRIORITY JOURNAL; PROTEIN DEGRADATION; PROTEIN LOCALIZATION; PROTEIN PROTEIN INTERACTION;

ANIMALIA;

EID: 0029989521     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.271.5256.1736     Document Type: Article

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30. Fly head extracts were prepared as described (12), TIM-specific antibodies were raised in rats against affinity-punfied glutathione-S-transferase fusion proteins expressing either residues 222 to 577 [antibodies (Ab) 305 and 307] or 1133 to 1389 (Ab 310) of TIM For protein immunoblots, Ab 307 was used, and for cytology Ab 305 was used The antibodies were prepared by HRP (Denver, PA) All blots were visualized by enhanced chemiluminescence (ECL, Amersham).
31. per ' flies were maintained in constant darkness for 4 days. At time zero a set of flies was pulsed with light ( - 8000 lux) for 1 hour, then returned to constant darkness Control flies and light-pulsed flies were harvested at O, 1 2 3, 5, 7, and 9 hours from the start ol pulse One group of flies was harvested immediately after 15 min of light exposure Head extracts and protein immunoblots were performed as in Fig 1A per ' flies were maintained for 4 days in LD12 12 and subdivided for use as controls (constant dark) or in 1 -hour light-pulse experiments as for per0 All molecular phase-resetting experiments were done at least four times (twice immunocytochemically and twice by protein immunoblotting) with similar results
32. cn bw flies were entrained to an LD cycle for at least 3 days and then transferred to constant darkness. Tenminute pulses of light (∼8000 lux) were administered at the indicated times For each time point, the average phase of the locomotor activity rhythms of 16 pulsed flies was compared to that of 16 untreated control flies. Activity rhythms were assessed continuously for several days in nonpulsed controls and after phase-resetting light pulses as described (4, 14). The new phase of the activity rhythm was usually evident in the record within 24 hours of light-pulse administration. Standard errors of the mean were derived from at least three independent experiments for each time point.
33. We thank J. Dunlap, J. Hall, P Model, M. Rosbash, A. Sehgal, and N. D Zinder for discussion and comments on the manuscript and M Rosbash and A Sehgal for communicating related unpublished observations Supported by the Howard Hughes Medical institute. M. W. Y. is an Investigator in the Howard Hughes Medical Institute.