Closing the circadian loop: CLOCK-induced transcription of its own inhibitors per and tim

Science

Volumn 280, Issue 5369, 1998, Pages 1599-1603

  Darlington, Thomas K ^a   Wager Smith, Karen ^a   Ceriani, M Fernanda ^a   Staknis, David ^a   Gekakis, Nicholas ^a   Steeves, Thomas D L ^a   Weitz, Charles J ^a   Takahashi, Joseph S ^a   Kay, Steve A ^a  

^a NONE

Author keywords

[No Author keywords available]

Indexed keywords

HELIX LOOP HELIX PROTEIN; PROTEIN INHIBITOR; TRANSCRIPTION FACTOR;

ANIMAL CELL; ARTICLE; BASE PAIRING; CIRCADIAN RHYTHM; CONTROLLED STUDY; DROSOPHILA; FEEDBACK SYSTEM; GENE EXPRESSION; MOUSE; NONHUMAN; PRIORITY JOURNAL; PROMOTER REGION; REPORTER GENE; SEQUENCE HOMOLOGY; TRANSACTIVATION; TRANSCRIPTION REGULATION;

ANIMALS; BASIC HELIX-LOOP-HELIX TRANSCRIPTION FACTORS; BIOLOGICAL CLOCKS; CELL LINE; CELL NUCLEUS; CIRCADIAN RHYTHM; DIMERIZATION; DROSOPHILA; DROSOPHILA PROTEINS; FEEDBACK; GENE EXPRESSION; HELIX-LOOP-HELIX MOTIFS; INSECT PROTEINS; NUCLEAR PROTEINS; PROMOTER REGIONS (GENETICS); RNA, MESSENGER; TRANS-ACTIVATION (GENETICS); TRANS-ACTIVATORS; TRANSCRIPTION FACTORS; TRANSFECTION;

EID: 0032486432     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.280.5369.1599     Document Type: Article

References (44)

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36. Expression plasmids contained the complete coding region of the indicated gene fused to the Drosophila actin 5C promoter in pAct (18). Reporter plasmids were as follows: per-luc, a 4.2-kb fragment of the period promoter fused to firefly luciferase (32); timluc, a 6.5-kb Bam HI-Sal I fragment of the timeless gene corresponding to the region upstream of the start of translation fused to firefly luciferase; and CME-lacZ, six Toll site 4 CMEs fused to lacZ (18). As a control for transfection efficiency, we used a plasmid containing the actin 5C promoter fused to Renilla luciferase in pRL-null (Promega). S2 cells in 12-well plates were transfected with Lipofectin (Gibco BRL) according to the manufacturer's recommendations. Each transfection contained 0.1 to 0.2 μg of each expression plasmid, 0.2 to 0.4 μg of reporter, 0.001 μg of control plasmid, and pAct to keep the amount of DNA per transfection constant. Cells were harvested 48 hours after transfection, and enzyme activity was measured with the Luciferase Assay, Dual-Luciferase Reporter Assay, and β-Galactosidase Enzyme Assay systems (Promega). For each sample, reporter activity was normalized to Renilla luc activity. Reporter activity was plotted relative to activity when cotransfected with empty expression vector pAct. Values are the mean ± SEM of two to four replicate experiments.
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38. Riboproprobes were generated with Ambion's Maxiscript kit (Austin, TX). Northern blots were performed by standard methods.
39. Frozen flies were vortexed and put through a tissue sieve. RNA was extracted from the fractions with RNAzolB (Tel-Test, Friendswood, TX). Ribonuclease (RNAse) protections were done on 100 μg of RNA with the RPAll kit (Ambion) according to the manufacturer's instructions. Positive control RNA and riboprobes were made with Ambion's Maxiscript kit.
40. Each multimer reporter consisted of four 18-bp elements containing the E box from either the per or tim promoter. Sequences shown in Fig. 2C are from the sense strand of the per promoter and the antisense strand of the tim promoter. To construct the reporters, the following synthetic oligonucleotides were annealed and inserted into a pGL3 vector (Promega) containing the hsp70 basal promoter: wild-type per multimer, 5′-GTACCGCCGCTCACGTGGCGAACC CTCGAGCCGCTCACGTGGCGAA-3′, 5′-CCCGC GAGCCGCTCACGTGGCGAACATTAATGCCGCT CACGTGGCGAACC-3′, 5′-CTCGCGGGTTCGCC ACGTGAGCGGCTCGAGGGTTCGCCACGTGA GCGGC-3′', 5′-TCGAGGTTCGCCACGTGAGCG GCATTAATGTTCGCCACGTGAGCGG-3′; mutant per multimer, 5′-GTACCGCCGCTCAGCTGGCGA ACCCTCGAGCCGCTCAGCTGGCGAA-3′. 5'-CC CGCGAGCCGCTCAGCTGGCGAACATTAATGCC GCTCAGCTGGCGAACC-3′, 5′-CTCGCGGGTT CGCCAGCTGAGCGGCTCGAGGGTTCGCCAG CTGAGCGGCG-3′, 5′-TCGAGGTTCGCCAGCTG AGCGGCATTAATGTTCGCCAGCTGAGCGG-3′; wild-type tim multimer, 5′-GTACCTGATTACACGTGAGCCGAAGAGATTGATTACACGTGAGCCG-3′, 5′-AGGCGGTTGATTACACGTGAGCCGAACTAGTTGATTACACGTGAGCCGAC-3′, 5′-AACCG CCTCGGCTCACGTGTAATCAATCTCTTCGGC TCACGTGTAATCAG-3′, 5′ -TCGAGTCGGCTC ACGTGTAATCAACTAGTTCGGCTCACGTGTA ATC-3′; mutant tim multimer, 5′-GTACCTGA TTACAGCTGAGCCGAAGAGATTGATTACAGCTG AGCCG-3′, 5′-AGGCGGTTGATTACAGCTGAG CCGAACTAGTTGATTACAGCTGAGCCGAC-3′, 5′-AACCGCCTCGGCTCAGCTGTAATCAATCTCTT CGGCTCAGCTGTAATCAG-3′, 5′ -TCGAGTCGGC TCAGCTGTAATCAACTAGTTCGGCTCAGCTGTAA TC-3′. Replicate experiments were performed, and a representative data set is shown.
41. Two-hybrid assays were carried out on 5-bromo-4-chloro-3-indolyl β-D-galactopyranoside (X-Gal) plates as described (33). For these assays, we used LEXA-dCLOCK(1-496) as bait, from which most of the COOH-temninal region was removed.
42. The indicated DNA fragments from the per gene clock control region were ligated into pBgl-lacZ, a one-hybrid reporter plasmid (25), and one-hybrid yeast reporter strains were constructed by insertion of recombinant pBgl-/acZ reporter plasmids into the ura3 locus of YPH 499 (34). The negative control strain was constructed identically with nonrecombinant pBgl-lacZ. One-hybrid reporter strains were transformed with the indicated expression plasmids, and transformants were patched onto X-Gal plates (33) for detection of β-galactosidase (β-Gal) activity. The 21-bp fragment is part of the 69-bp circadian-regulated region (10) and contained the E-box with 8 bp of 5′ and 7 bp of 3′ flanking sequences. For the 21-bp fragment with the mutated E-box, the E-box sequence was scrambled by means of a random number table. E-box mutant: 5′-ATTCGC.
43. Plasmids expressing per and tim from the actin 5C promoter in pAct were constructed. Transfections and assays were performed as described (35) with minor modifications. For each well, expression plasmids were used at 0.001 μg for dCLOCK and 0.01 μg for PER and TIM. An additional plasmid with the hsp70 promoter driving lacZ was used at 0.1 μg per well to control for transfection efficiency. For each sample, activity was normalized to β-Gal activity. For each reporter, activity was normalized to activity when transfected with pAct. The value for dCLOCK-activated wild-type reporter was set to one. Replicate experiments were performed, and a representative data set is shown.
44. We thank M. Myers, L. Saez, and M. Young for the tim sequence and clones; T. L. Schwartz for the head cDNA library; J. Park and J. Hall for in situ chromosomal mapping of dclock; R. Stanewsky, S. Crews, and P. Hardin for various clones; J. Kreps, R. Raman, and C. Andersson for their generous help; and A. McLachlan and P. Ghazal for helpful discussions. This work was supported by National Institutes of Mental Health grant MH-51573 and the NSF Center for Biological Timing (S.A.K.), the Pew Foundation (fellowship to M.F.C.), and an NSF and McKnight Scholars Award (C.J.W.). J.S.T. is an Investigator of the Howard Hughes Medical Institute.