Real-Time Luminescence Reporting of Circadian Gene Expression in Mammals

Yamazaki, Shin; Takahashi, Joseph S.

doi:10.1016/s0076-6879(05)93012-7

Cited by 172 publications

(157 citation statements)

References 28 publications

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“…The catalytic subunits of DNA-dependent protein kinase (DNA-PKcs) WT and KO MEF were grown in DMEM supplemented with 10% FBS, 100 units/ml penicillin, 100 mg/ml streptomycin and cultured at 37°C in a humidified incubator with 5% CO 2 . Cells were synchronized with 100 nM dexamethasone, and real time bioluminescence was recorded as described (26). The rhythms were analyzed by performing base-line subtraction followed by sine curve fitting using the Lumicycle software (Actimetrics).…”

Section: Methodsmentioning

confidence: 99%

Phosphorylation of the Cryptochrome 1 C-terminal Tail Regulates Circadian Period Length

Gao

Yoo

Lee

et al. 2013

Journal of Biological Chemistry

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Background: Cryptochromes (CRYs) are transcriptional repressors that are critical components of the circadian clock. Results: We have identified a phosphorylation site in the CRY1 tail that is negatively regulated by the DNA repair enzyme DNA-dependent protein kinase. Conclusion: Phosphorylation of CRY1 on Ser-588 increases its half-life and lengthens the circadian period. Significance: The C-terminal tail of CRY1 modulates period length.

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Section: Methodsmentioning

confidence: 99%

Phosphorylation of the Cryptochrome 1 C-terminal Tail Regulates Circadian Period Length

Gao

Yoo

Lee

et al. 2013

Journal of Biological Chemistry

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“…Fortyeight hours later, growth media (Dulbecco's modified Eagle medium; DMEM; Gibco) containing 5% fetal bovine serum (FBS) were replaced with recording media (DMEM no phenol red; Sigma) with sodium bicarbonate (350 mg/L; Sigma), Hepes (10 mM; Sigma), 5% FBS (FPJ21922; HyClone), and luciferin (0.1 mM; Promega) and the luminescence signal was measured. The detailed methods for real-time measurement of luminescence have been described (41). Bioluminescence was measured using a photon-counting head (H6240; Hamamatsu) in a temperature-controlled environmental room (Environmental Growth Chambers).…”

Section: Methodsmentioning

confidence: 99%

Circadian-independent cell mitosis in immortalized fibroblasts

Yeom

Pendergast

Ohmiya

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

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Two prominent timekeeping systems, the cell cycle, which controls cell division, and the circadian system, which controls 24-h rhythms of physiology and behavior, are found in nearly all living organisms. A distinct feature of circadian rhythms is that they are temperaturecompensated such that the period of the rhythm remains constant (∼24 h) at different ambient temperatures. Even though the speed of cell division, or growth rate, is highly temperature-dependent, the cell-mitosis rhythm is temperature-compensated. Twenty-fourhour fluctuations in cell division have also been observed in numerous species, suggesting that the circadian system is regulating the timing of cell division. We tested whether the cell-cycle rhythm was coupled to the circadian system in immortalized rat-1 fibroblasts by monitoring cell-cycle gene promoter-driven luciferase activity. We found that there was no consistent phase relationship between the circadian and cell cycles, and that the cell-cycle rhythm was not temperature-compensated in rat-1 fibroblasts. These data suggest that the circadian system does not regulate the cell-mitosis rhythm in rat-1 fibroblasts. These findings are inconsistent with numerous studies that suggest that cell mitosis is regulated by the circadian system in mammalian tissues in vivo. To account for this discrepancy, we propose two possibilities: (i) There is no direct coupling between the circadian rhythm and cell cycle but the timing of cell mitosis is synchronized with the rhythmic host environment, or (ii) coupling between the circadian rhythm and cell cycle exists in normal cells but it is disconnected in immortalized cells.O rganization of physiology and behavior into specific time domains is a fundamental property of nearly all living organisms. Anticipation of periodic changes in the environment presumably increased survival and reinforced the development of endogenous circadian oscillators (1). Because cell division is critical to the survival of unicellular organisms and the integrity of DNA is susceptible to UV irradiation, the progression of the cell cycle was probably also strongly affected by daily changes in the environment. Indeed, multiple studies have measured diurnal fluctuations in cell division such that mitosis occurs at a specific time of day in numerous species ranging from unicellular organisms (2) to humans (3-5). These data suggest that the circadian and cell cycles may be coupled in vivo.Circadian rhythms are self-sustained oscillations in physiology and behavior with endogenous periods of ≈24 h that can be synchronized, or entrained, to environmental cues such as the light/dark cycle or temperature (6). In mammals, the master circadian clock is located in the suprachiasmatic nuclei (SCN) of the anterior hypothalamus. Genes that are important for circadian timekeeping are expressed not only in the SCN but also in many peripheral tissues, including fibroblasts (7-11). Immortalized embryonic fibroblasts exhibit circadian rhythms of gene expression (12) and, using singlecell imaging...

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“…We were surprised that this basic environmental variable, which has important implications for setting upper IPI cut-offs, was so poorly controlled. quickly, particularly when they are generated in the periphery (27) as song rhythms are likely to be (28). This may be reflected in the observation that for the 25 most vigorous Canton-S songs we selected (see below), 19 of 25 songs had higher mean IPIs in the 1st compared with the 45th minute (34.98 vs. 33.92 ms, paired t, P = 0.0039, distribution χ 2 , P = 0.009).…”

Section: Significancementioning

confidence: 99%

Failure to reproduce period -dependent song cycles in Drosophila is due to poor automated pulse-detection and low-intensity courtship

Kyriacou

Green

Piffer

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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Stern has criticized a body of work from several groups that have independently studied the so-called "Kyriacou and Hall" courtship song rhythms of male Drosophila melanogaster, claiming that these ultradian ∼60-s cycles in the interpulse interval (IPI) are statistical artifacts that are not modulated by mutations at the period (per) locus [Stern DL (2014) BMC Biol 12:38]. We have scrutinized Stern's raw data and observe that his automated song pulse-detection method identifies only ∼50% of the IPIs found by manual (visual and acoustic) monitoring. This critical error is further compounded by Stern's use of recordings with very little song, the large majority of which do not meet the minimal song intensity criteria which Kyriacou and Hall used in their studies. Consequently most of Stern's recordings only contribute noise to the analyses. Of the data presented by Stern, only per L and a small fraction of wild-type males sing vigorously, so we limited our reanalyses to these genotypes. We manually reexamined Stern's raw song recordings and analyzed IPI rhythms using several independent time-series analyses. We observe that per L songs show significantly longer song periods than wildtype songs, with values for both genotypes close to those found in previous studies. These per-dependent differences disappear when the song data are randomized. We conclude that Stern's negative findings are artifacts of his inadequate pulse-detection methodology coupled to his use of low-intensity courtship song records.D uring courtship, the Drosophila melanogaster male vibrates his wing toward the female and produces a series of pulses and hums (1). The pulses have a variable interpulse interval (IPI), which ranges from 15 to 80 ms but usually averages between 30 and 40 ms, whereas sympatric Drosophila simulans mean IPIs vary from 45 to 80 ms depending on the strain (2, 3). In 1980, in these pages, the first of a series of studies by Kyriacou, Hall, and their collaborators revealed that superimposed on these IPIs was a low-amplitude oscillation of about 60 s in D. melanogaster and 40 s in D. simulans (4-12). Furthermore, in D. melanogaster, these cycles were modulated in a predictable fashion by the circadian rhythm period (per) mutations (13): per L males with long 29-h circadian cycles also sang with long ∼80-s song cycles, whereas circadian arrhythmic per 01 males showed a corresponding song phenotype (2, 4, 9, 10). These cycles were shown to have functional significance in playback experiments in which females were shown to be most responsive to both their species-specific IPI and cycle (2, 14-16).The work of Kyriacou, Hall, and collaborators was performed in the late 1970s and 1980s using extremely laborious analog technology, so to attain any kind of throughput, IPIs were binned into consecutive 10-s intervals and a mean IPI calculated on a minimum of 10 IPIs (2, 4-6, 8). Initially, sine/cosine functions were fitted to the mean IPIs (2, 4-6, 8), and this was later complemented by spectral analyses, with both types of statisti...

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Real-Time Luminescence Reporting of Circadian Gene Expression in Mammals

Cited by 172 publications

References 28 publications

Phosphorylation of the Cryptochrome 1 C-terminal Tail Regulates Circadian Period Length

Phosphorylation of the Cryptochrome 1 C-terminal Tail Regulates Circadian Period Length

Circadian-independent cell mitosis in immortalized fibroblasts

Failure to reproduce period -dependent song cycles in Drosophila is due to poor automated pulse-detection and low-intensity courtship

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