A 69 bp circadian regulatory sequence (CRS) upstream of the per gene is sufficient to drive circadian transcription, mediate proper spatial expression, and rescue behavioral rhythmicity in per01 flies. Within the CRS, an E-box is required for transcriptional activation by two basic-helix-loop-helix (bHLH) PERARNT-SIM (PAS) transcription factors, dCLOCK (dCLK) and CYCLE (CYC). To define sequences within the CRS that are required for spatial expression, circadian expression, and behavioral rhythmicity, a series of mutants that alter blocks of 3 to 12 nucleotides across the entire CRS were used to drive lacZ or per expression in vivo. As expected, the E-box within the CRS is necessary for high-level expression and behavioral rhythmicity, but sequences outside the E-box are also required for transcriptional activation, proper spatial expression, and behavioral rhythmicity. These results indicate that the dCLK-CYC target site extends beyond the E-box and that factors other than dCLK and CYC modulate per transcription.
Allada R, White NE, So WV, Hall JC, and Rosbash M (1998) A mutant Drosophila homolog of mammalian Clock disrupts circadian rhythms and transcription of period and timeless. Cell93:791-804.
2.
Blau J and Young MW (1999) Cycling vrille expression is required for a fully functional Drosophila clock. Cell99:661-671.
3.
Cheng Y and Hardin PE (1998) Drosophila photoreceptors contain an autonomous circadian oscillator that can function without period mRNAcycling. J Neurosci18:741-750.
4.
Curtin KD, Huang ZJ, and Rosbash M (1995) Temporally regulated entry of the Drosophila period protein contributes to the circadian clock. Neuron14:365-372.
5.
Darlington TK, Lyons LC, Hardin PE, and Kay SA(2000) The period E-box is sufficient to drive circadian oscillation of transcription in vivo. J Biol Rhythms15:462-471.
6.
Darlington TK, Wager-Smith K, Ceriani MF, Staknis D, Gekakis N, Steeves TDL, Weitz CJ, Takahashi JS, and Kay SA (1998) Closing the circadian feedback loop: CLOCK induced transcription of its own inhibitors, period and timeless. Science280:1599-1603.
7.
Dunlap JC (1999) Molecular bases for circadian clocks. Cell96:271-290.
8.
Edery I, Zwiebel LJ, Dembinska ME, and Rosbash M (1994) Temporal phosphorylation of the Drosophila period protein. Proc Natl Acad Sci U S A91:2260-2264.
9.
Ewer J, Hamblen-Coyle M, Rosbash M, and Hall JC (1990) Requirement for period gene expression in the adult and not during development for locomotor activity rhythms of imaginal Drosophila melanogaster. J Neurogenet7:31-73.
10.
Gekakis N, Staknis D, Nguyen HB, Davis FC, Wilsbacher LD, King DP, Takahashi JS, and Weitz CJ (1998) Role of the CLOCK protein in the mammalian circadian mechanism. Science280:1564-1569.
11.
Hamblen MJ, White NE, Emery PT, Kaiser K, and Hall JC (1998) Molecular and behavioral analysis of four periodmutants in Drosophila melanogaster encompassing extreme short, novel long, and unorthodox arrhythmic types. Genetics149:165-178.
12.
Hamblen M, Zehring WA, Kyriacou CP, Reddy P, Yu Q, Wheeler DA, Zwiebel LJ, Konopka RJ, Rosbash M, and Hall JC (1986) Germ-line transformation involving DNA from the period locus in Drosophila melanogaster: Overlapping genomic fragments that restore circadian and ultradian rhythmicity to per and per mutants. J Neurogenet3:249-291.
13.
Hao H, Allen DL, and Hardin PE (1997) A circadian enhancer mediates PER-dependent mRNA cycling in Drosophila. Mol Cell Biol17:3687-3693.
14.
Hao H, Glossop NRJ, Lyons L, Qiu J, Morrish B, Cheng Y, Helfrich-Forster C, and Hardin PE (1999) The 69 bp circadian regulatory sequence (CRS) mediates per-like developmental, spatial, and circadian expression and behavioral rescue in Drosophila. J Neurosci19:987-994.
15.
Hardin PE, Hall JC, and Rosbash M (1990) Feedback of the Drosophila period gene product on circadian cycling of its messenger RNA levels. Nature342:536-540.
16.
Hardin PE, Hall JC, and Rosbash M (1992) Circadian oscillations in period gene mRNA levels are transcriptionally regulated. Proc Natl Acad Sci U S A89:11711-11715.
17.
Hardin SH, Jones LB, Homayouni R, and McCollum JC (1996) Octamer primed cycle sequencing: Design of an optimized primer library. Genome Res6:545-550.
18.
Hogenesch JB, Gu Y-Z, Jain S, and Bradfield CA (1998) The basic-helix-loop-helix-PAS orphan MOP3 forms transcriptionally active complexes with circadian and hypoxia factors. Proc Natl Acad Sci U S A95:5474-5479.
19.
Jin X, Shearman LP, Weaver DR, Zylka MJ, DeVries GJ, and Reppert SM (1999) A molecular mechanism regulating rhythmic output from the suprachiasmatic circadian clock. Cell96:57-68.
20.
Kloss B, Price JL, Saez L, Balu J, Rothenfluh A, Wesley C, and Young MW (1998) The Drosophila clock gene double-timeencodes a protein closely related to human casein kinase I[.epsilon]. Cell94:97-107.
21.
Lee C, Bae K, and Edery I (1999) PER and TIM inhibit the DNA binding activity of a dCLOCK-CYC/dBMAL1 heterodimer without disrupting formation of the heterodimer: Abasis for circadian transcription. Mol Cell Biol19:5316-5325.
22.
Liu X, Lorenz LJ, Yu Q, Hall JC, and Rosbash M (1988) Spatial and temporal expression of the period gene in Drosophila melanogaster. Genes Dev2:228-238.
23.
Liu X, Zwiebel LJ, Hinton D, Benzer S, Hall JC, and Rosbash M (1992) The period gene encodes a predominantly nuclear protein in adult Drosophila. J Neurosci12:2735-2744.
24.
Monsma SA, Ard R, Lis JT, and Wolfner MF (1988) Localized heat-shock induction in Drosophila melanogaster. J Exp Zool247:279-284.
25.
Price JL, Blau J, Rothenfluh A, Abodeely M, Kloss B, and Young MW (1998) double-time is a new Drosophila clock gene that regulates PERIOD protein accumulation. Cell94:83-95.
26.
Price JL, Dembinska ME, Young MW, and Rosbash M (1995) Suppression of PERIOD protein abundance and circadian cycling by the Drosophila clock mutation timeless. EMBO J14:4044-4049.
27.
Rutila JE, Suri V, Le M, So WV, Rosbash M, and Hall JC (1998) CYCLE is a second bHLH-PAS clock protein essential for circadian rhythmicity and transcription of Drosophila period and timeless. Cell93:805-814.
28.
Sehgal A, Price JL, Man B, and Young MW (1994) Loss of circadian behavioral rhythms and per RNA oscillations in the Drosophila mutant timeless. Science263:1603-1606.
29.
Sehgal A, Rothenfluh-Hilfiker A, Hunter-Ensor M, Chen Y, Myers MP, and Young MW (1995) Rhythmic expression of timeless: Abasis for promoting circadian cycles in periodgene autoregulation. Science270:808-810.
30.
Vallette F, Mege E, Reiss A, and Adesnik M (1989) Construction of mutant and chimeric genes using the polymerase chain reaction. Nucleic Acids Res17:723-733.
31.
Vosshall LB, Price JL, Sehgal A, Saez L, and Young MW (1994) Block in nuclear localization of period protein by a second clock mutation, timeless. Science263:1606-1609.
32.
Wharton KA and Crews ST (1993) CNS midline enhancers of the Drosophila slit and Toll genes. Mech Dev40:141-154.
33.
Zerr DM, Hall JC, Rosbash M, and Siwicki KK (1990) Circadian fluctuations of period protein immunoreactivity in the CNS and the visual system of Drosophila. J Neurosci10:2749-2762.
34.
Zwiebel LJ, Hardin PE, Liu X, Hall JC, and Rosbash M (1991) A post-transcriptional mechanism contributes to circadian cycling of a per-b-galactose fusion protein. Proc Natl Acad Sci U S A88:3882-3886.
