Clock Gene mRNA Research Articles

The resetting of the circadian rhythm by Prostaglandin J 2 is distinctly phase-dependent

The circadian rhythm can be reset by a variety of substances. Prostaglandin J 2 (PGJ 2) is one such substance and resets the circadian rhythm in fibroblasts. In our current study, we examined the phase-dependent phase shift following PGJ 2 treatment using a real-time luciferase luminescence monitoring system. In the phase response curves, we observed 12 h differences in the times of peaks in comparison with the same analysis for forskolin. Quantification of clock gene mRNAs following PGJ 2 administration additionally revealed a rapid decrease in the Per1, Rev-erbAα and Dbp levels. Our current findings thus suggest that PGJ 2 resets the peripheral circadian clock via a mechanism that is distinct from that used by forskolin (FK).

Modulation of metabolic and clock gene mRNA rhythms by pineal and retinal circadian oscillators

Avian circadian organization involves interactions between three neural pacemakers: the suprachiasmatic nuclei (SCN), pineal, and retina. Each of these structures is linked within a neuroendocrine loop to influence downstream processes and peripheral oscillations. However, the contribution of each structure to drive or synchronize peripheral oscillators or circadian outputs in avian species is largely unknown. To explore these interactions in the chick, we measured 2-deoxy[ 14C]-glucose (2DG) uptake and mRNA expression of the chick clock genes bmal1, cry1, and per3 in three brain areas and in two peripheral organs in chicks that underwent pinealectomy, enucleation, or sham surgery. We found that 2DG uptake rhythms damp under constant darkness in intact animals, while clock gene mRNA levels continue to cycle, demonstrating that metabolic rhythms are not directly driven by clock gene transcription. Moreover, 2DG rhythms are not phase-locked to rhythms of clock gene mRNA. However, pinealectomy and enucleation had similar disruptive effects on both metabolic and clock gene rhythms, suggesting that both of these oscillators act similarly to reinforce molecular and physiological rhythms in the chicken. Finally, we show that the relative phasing of at least one clock gene, cry1, varies between central and peripheral oscillators in a tissue specific manner. These data point to a complex, differential orchestration of central and peripheral oscillators in the chick, and, importantly, indicate a disconnect between canonical clock gene regulation and circadian control of metabolism.

Melatonin and the circadian entrainment of metabolic and hormonal activities in primary isolated adipocytes

The aim of this work was to investigate the effect of the in vitro circadian-like exposure to melatonin [in the presence or absence of insulin (Ins)] on the metabolism and clock gene expression in adipocytes. To simulate the cyclic characteristics of the daily melatonin profile, isolated rat adipocytes were exposed in a circadian-like pattern to melatonin added to the incubating medium for 12 hr (mimicking the night), followed by an equal period without melatonin (mimicking the day) combined or not with Ins. This intermittent incubation was interrupted when four and a half 24-hr cycles were fulfilled. At the end, either during the induced night (melatonin present) or the induced day (melatonin absent), the rates of lipolysis and D-[U-(14)C]-glucose incorporation into lipids were estimated, in addition to the determination of lipogenic [glucose-6-phosphate dehydrogenase and fatty acid synthase (FAS)] and lipolytic (hormone sensitive lipase) enzymes and clock gene (Bmal-1b, Clock, Per-1 and Cry-1) mRNA expression. The leptin release was also measured. During the induced night, the following effects were observed: an increase in the mRNA expression of Clock, Per-1 and FAS; a rise in lipogenic response and leptin secretion; and a decrease in the lipolytic activity. The intermittent exposure of adipocytes to melatonin temporally and rhythmically synchronized their metabolic and hormonal function in a circadian fashion, mimicking what is observed in vivo in animals during the daily light-dark cycle. Therefore, this work helps to clarify the physiological relevance of the circadian pattern of melatonin secretion and its interactions with Ins, contributing to a better understanding of the adipocyte biology.

Molecular characterization of the circadian clock genes in the bean bug, Riptortus pedestris, and their expression patterns under long- and short-day conditions

Although the molecular mechanisms and the diversity of insect circadian clocks have been well investigated in holometabolous insects, hemimetabolous insects have received little attention. In the present study, we isolated the circadian clock genes, period ( per), cycle ( cyc), vrille ( vri), and mammalian-type cryptochrome ( cry-m) from the bean bug Riptortus pedestris. This is the first report of vri and cry-m in hemimetabolous insects. All of the genes showed high similarities to respective homologous genes in other insects. The discovery of cry-m in R. pedestris indicates that the clockwork of hemimetabolous insects is similar to that in insects having CRY-m, including the monarch butterfly Danaus plexippus and the honey bee Apis mellifera, and not to insects lacking it, such as Drosophila melanogaster. Real-time PCR showed that mRNAs of these circadian clock genes exhibited extremely weak diel oscillations at day 9 in the head of R. pedestris, and their expression levels under long- and short-day conditions were nearly identical. In addition, expression levels of per mRNA were almost stable from days 0 to 15 under both photoperiodic conditions. The difference between long-day and short-day conditions in the mRNA level seems too small to distinguish photoperiodic conditions clearly. These results suggest that transcriptional regulations of circadian clock genes would not play an important role in the diapause programming in R. pedestris.

Clock Gene Expression in the Murine Gastrointestinal Tract: Endogenous Rhythmicity and Effects of a Feeding Regimen

Based on observations that the gastrointestinal tract is subject to various 24-hour rhythmic processes, it is conceivable that some of these rhythms are under circadian clock gene control. We hypothesized that clock genes are present in the gastrointestinal tract and that they are part of a functional molecular clock that coordinates rhythmic physiologic functions. The effects of timed feeding and vagotomy on temporal clock gene expression (clock, bmal1, per1-3, cry1-2) in the gastrointestinal tract and suprachiasmatic nucleus (bmal, per2) of C57BL/6J mice were examined using real-time polymerase chain reaction and Western blotting (BMAL, PER2). Colonic clock gene localization was examined using immunohistochemistry (BMAL, PER1-2). Clock immunoreactivity was observed in the myenteric plexus and epithelial crypt cells. Clock genes were expressed rhythmically throughout the gastrointestinal tract. Timed feeding shifted clock gene expression at the RNA and protein level but did not shift clock gene expression in the central clock. Vagotomy did not alter gastric clock gene expression compared with sham-treated controls. The murine gastrointestinal tract contains functional clock genes, which are molecular core components of the circadian clock. Daytime feeding in nocturnal rodents is a strong synchronizer of gastrointestinal clock genes. This synchronization occurs independently of the central clock. Gastric clock gene expression is not mediated through the vagal nerve. The presence of clock genes in the myenteric plexus and epithelial cells suggests a role for clock genes in circadian coordination of gastrointestinal functions such as motility, cell proliferation, and migration.

Induction of Drosophila Behavioral and Molecular Circadian Rhythms by Temperature Steps in Constant Light

In constant light, where Drosophila rhythms are normally disrupted, temperature cycles induce circadian rhythms at both the molecular and behavioral level. The authors investigated the process by which the thermoperiod induces the rhythms using temperature steps. A 10 degrees C temperature step-up induced a single locomotor activity peak ca 9 h after the temperature transition, whereas a 10 degrees C step-down induced a strong activity peak ca 24 h after the transition, and the peak recurred for several cycles, suggesting that the underlying clock is reset. Arrhythmic per(01) , tim( 01) , dClk(Jrk) , and cyc(01) mutant flies failed to show the rhythm after the step-down, suggesting that per, tim, dClk, and cyc are necessary for the step-down-induced rhythm. After the step-up, per(01) flies exhibited an activity peak similar to that of wild-type flies, suggesting that the peak can be induced by the step-up in absence of PER. mRNA levels of per, tim , dClk, vri, and Pdp1epsilon were changed in response to the temperature steps, but the changes differed depending on the direction of temperature steps, suggesting that steps-up and steps-down have different roles in the initiation of the oscillation. Probably, alternating 12-h temperature steps-up and steps-down will induce opposite changes in mRNA levels of clock genes, eventually producing stable molecular oscillations. Although TIM shows responses to temperature consistent with the changes of its mRNA, this is not the case for PER, consistent with posttranscriptional regulation. Changes of the mRNA levels were significantly altered but still observed in per( 01) flies but not observed in dClk(Jrk) flies, except for per mRNA. This suggests that dCLK is involved in the temperature-induced changes in the levels of most clock gene mRNA but that per is regulated via a different mechanism.

Circadian Variations in Clock Gene Expression of Human Bone Marrow CD34+ Cells

Time-dependent variations in clock gene expression have recently been observed in mouse hematopoietic cells, but the activity of these genes in human bone marrow (BM) has so far not been investigated. Since such data can be of considerable clinical interest for monitoring the dynamics in stem/progenitor cells, the authors have studied mRNA expression of the clock genes hPer1 , hPer2, hCry1, hCry2, hBmal1, hRev-erb alpha, and hClock in human hematopoietic CD34-positive (CD34( +)) cells. CD34(+) cells were isolated from the BM samples obtained from 10 healthy men at 6 times over 24 h. In addition, clock gene mRNA expression was analyzed in the whole BM in 3 subjects. Rhythms in serum cortisol, growth hormone, testosterone, and leukocyte counts documented that subjects exhibited standardized circadian patterns. All 7 clock genes were expressed both in CD34(+) cells and the whole BM, with some differences in magnitude between the 2 cell populations. A clear circadian rhythm was shown for hPer1, hPer2, and hCry2 expression in CD34(+) cells and for hPer1 in the whole BM, with maxima from early morning to midday. Similar to mouse hematopoietic cells, h Bmal1 was not oscillating rhythmically. The study demonstrates that clock gene expression in human BM stem/progenitor cells may be developmentally regulated, with strong or weaker circadian profiles as compared to those reported in other mature tissues.

Influence of Temperature on the Liver Circadian Clock in the Ruin Lizard Podarcis sicula

Reptiles represent an interesting animal model to investigate the influence of temperature on molecular circadian clocks. The ruin lizard Podarcis sicula lives in a continental climate and it is subjected to wide range of environmental temperatures during the course of the year. As consequence, ruin lizard daily activity pattern includes either the hibernation or periods of inactivity determined by hypothermia. Here we showed the rhythmic expression of two clock genes, lPer2 and lClock, in the liver of active lizards exposed to summer photo-thermoperiodic conditions. Interestingly, the exposition of lizards to hypothermic conditions, typical of winter season, induced a strong dampening of clock genes mRNA rhythmicity with a coincident decrease of levels. We also examined the qualitative and quantitative distribution of lPER2 and lCLOCK protein in different cellular compartments during the 24-h cycle. In the liver of active lizards both proteins showed a rhythmic expression profile in all cellular compartments. After 3 days at 6 degrees C, some temporal fluctuations of the lCLOCK and lPER2 are still detectable, although, with some marked modifications in respect to the values detected in the liver of active lizards. Besides demonstrating the influence of low temperature on the lizard liver circadian oscillators, present results could provide new essential information for comparative studies on the influence of temperature on the circadian system across vertebrate classes.

Development of the rhythmic melatonin secretion in the embryonic chicken pineal gland

In order to elucidate details on the development of the circadian clock, the effects of light on the in vitro melatonin (MT) release and the presence of mRNAs of several clock genes in the embryonic chicken pineal gland were investigated. Chicken embryos of various developmental stages were exposed to stimuli of light in vitro in dynamic, four day long bioassay (perifusion). MT secretion and mRNA levels of Cry1, Cry2, Clock and Bmal2 clock genes were determined. Our conclusions: (1) environmental illumination modified MT secretion from explanted embryonic pineal glands as early as on the 13th embryonic day, (2) daily rhythm of MT release develops between embryonic days 16 and 18 under periodic environmental illumination. (3) Chicken Cry1, Cry2, Clock and Bmal2 clock gene mRNAs were also detected in glands of animals of 15th embryonic day. Although both MT secretion and clock genes have been developed by then, the circadian MT rhythm appears first on the 17th embryonic day. Either the mechanisms coupling the clock with the melatonin output or the synchronization of the individual pinealocytes develop around this age. Rhythmic MT release in the embryonic chicken pineal gland evolves only if the egg is exposed to rhythmic environmental stimuli.

Circadian Rhythms in the CNS and Peripheral Clock Disorders: Function of Clock Genes: Influence of Medication for Bronchial Asthma on Circadian Gene

Bronchial asthma is a chronic inflammatory disorder of the airways, in which inflammation causes bronchial hyper-responsiveness and flow limitation in the presence of various stimuli. Pulmonary function in asthmatic patients frequently deteriorates between midnight and early morning, which has suggested a role for chronotherapy. Although relationships between bronchial asthma and the function of clock genes remain unclear, some medications given for asthma such as glucocorticoids or β2-adrenoceptor agonists may influence clock genes in vivo. In our studies of clock gene mRNA expressions in human bronchial epithelial cells in vitro and peripheral blood cells in vivo, we demonstrated that glucocorticoid or β2-adrenoceptor agonist treatment strongly induced human Per1 mRNA expression both in vitro and in vivo. Human peripheral blood cells provide a useful indication of peripheral clock gene mRNA expression in vivo.

Lack of Food Anticipation in Per2 Mutant Mice

Predicting time of food availability is key for survival in most animals. Under restricted feeding conditions, this prediction is manifested in anticipatory bouts of locomotor activity and body temperature. This process seems to be driven by a food-entrainable oscillator independent of the main, light-entrainable clock located in the suprachiasmatic nucleus (SCN) of the hypothalamus . Although the SCN clockwork involves self-sustaining transcriptional and translational feedback loops based on rhythmic expression of mRNA and proteins of clock genes , the molecular mechanisms responsible for food anticipation are not well understood. Period genes Per1 and Per2 are crucial for the SCN's resetting to light . Here, we investigated the role of these genes in circadian anticipatory behavior by studying rest-activity and body-temperature rhythms of Per1 and Per2 mutant mice under restricted feeding conditions. We also monitored expression of clock genes in the SCN and peripheral tissues. Whereas wild-type and Per1 mutant mice expressed regular food-anticipatory activity, Per2 mutant mice did not show food anticipation. In peripheral tissues, however, phase shifts of clock-gene expression in response to timed food restriction were comparable in all genotypes. In conclusion, a mutation in Per2 abolishes anticipation of mealtime, without interfering with peripheral synchronization by feeding cycles.

Circadian clocks regulate adenylyl cyclase activity rhythms in human RPE cells

Genes and components of the circadian clock may represent relevant drug targets for diseases involving circadian dysfunctions. By exploiting an established cell line derived from human retinal pigment epithelium (HRPE), the cell constituting the blood-retinal barrier that is essential to maintain the visual functions of the sensorineural retina, we showed serum-shock induction of rhythmic changes in forskolin-evoked adenylyl cyclase (AC) activity. In the presence of Ca 2+ and protein kinase A, the forskolin-induced AC activity is significantly, but not completely inhibited, suggesting the involvement of both Ca 2+-sensitive and Ca 2+-insensitive AC isoforms in the regulation of circadian rhythmicity in these cells. Semi-quantitative RT-PCR showed circadian profile in the expression of three AC isoforms, the Ca 2+-inhibitable AC5 and AC6 and the Ca 2+-insensitive AC7, and the clock genes hPer1 and hPer2. Our results demonstrate for the first time circadian rhythmicity in a human cell line, identifying the isoforms involved in the circadian profile of AC activity and showing a rhythmicity of the clock gene mRNA expression in these cells. Therefore, the results reported here provide evidence for an intertwine between AC/[Ca 2+] i signalling pathways and Per genes in the HRPE circadian clockwork.

Essential Role of 3′-Untranslated Region-mediated mRNA Decay in Circadian Oscillations of Mouse Period3 mRNA

Daily oscillations in mRNA levels are a general feature of most clock genes. Although mRNA oscillations largely depend on transcriptional regulation, it has been suggested that post-transcriptional controls also contribute to mRNA oscillations in Drosophila. Currently, however, there is no direct evidence for post-transcriptional regulation of mammalian clock genes. To investigate the roles of post-transcriptional regulations, we focused on the 3'-untranslated region (3'-UTR) of mouse Period3 (mPer3) mRNA, one of the clock genes. Insertion of the entire mPer3 3'-UTR downstream of a reporter gene resulted in a dramatic decrease in mRNA stability. Deletion and point mutation analyses led to the identification of critical sequences responsible for mRNA decay. To explore the effects of the mPer3 3'-UTR-mediated mRNA decay on circadian oscillations, we established NIH3T3 stable cell lines that express luciferase mRNA with wild-type or mutant mPer3 3'-UTR. Interestingly, a stabilizing mutation of 3'-UTR induced a significant alteration in the oscillation profile of luciferase mRNA. Above all, the peak time, during which the mRNAs reached their highest levels, was significantly delayed (for 12 h). In addition, the luciferase mRNA level with mutant 3'-UTR began to increase earlier than that in the presence of wild-type 3'-UTR. Consequently, luciferase mRNA with mutant 3'-UTR displayed oscillation patterns with a prolonged rising phase. Our results indicate that mPer3 3'-UTR-mediated mRNA decay plays an essential role in mRNA cycling and provide direct evidence for post-transcriptional control of circadian mRNA oscillations.

Identification of clock as a mechanosensitive gene by large-scale DNA microarray analysis: downregulation in osteoarthritic cartilage

The chondrocytes of cartilage have so far been reported to include three mechanoresponsive genes: cartilage matrix protein (CMP, matrilin-1), type X collagen, and Indian hedgehog (Ihh). In fact, all of the genes identified in these chondrocytes are associated solely with mechanical stress. In this study we examined the how mechanical stress influenced the patterns of gene expression of chondrocytes in three-dimensional (3D) sponges in order to better understand the mechanisms that control the chondrocyte phenotype during the development of osteoarthritis. We cultured mouse chondrocytes in 3D sponge as a model of mechanical stress and isolated total RNA for a large-scale DNA microarray analysis covering 12,000 genes. We analyzed the pattern of gene expression in relation to gene localization in cellular components such as the cytoplasm, cytoskeleton, and nucleus. Immunoblotting of osteoarthritis cartilage were performed using polyclonal anti-clock antibody. The biological rhythm of mRNA of clock and clock-related genes was analyzed by real-time polymerase chain reaction (PCR). Clock gene was confirmed by real-time PCR to validate the microarray data. Other clock-related genes such as Per1 and Per2 were also expressed in chondrocytes, exhibiting biological rhythm after serum shock. The large-scale DNA microarray indicated that clock and other genes functionally associated with mechanical stress play an essential role in regulating the biological rhythm of chondrocyte metabolism in osteoarthritis cartilage.

Identification of clock as a mechanosensitive gene by large-scale DNA microarray analysis: downregulation in osteoarthritic cartilage

The chondrocytes of cartilage have so far been reported to include three mechanoresponsive genes: cartilage matrix protein (CMP, matrilin-1), type X collagen, and Indian hedgehog (Ihh). In fact, all of the genes identified in these chondrocytes are associated solely with mechanical stress. In this study we examined the how mechanical stress influenced the patterns of gene expression of chondrocytes in three-dimensional (3D) sponges in order to better understand the mechanisms that control the chondrocyte phenotype during the development of osteoarthritis. We cultured mouse chondrocytes in 3D sponge as a model of mechanical stress and isolated total RNA for a large-scale DNA microarray analysis covering 12000 genes. We analyzed the pattern of gene expression in relation to gene localization in cellular components such as the cytoplasm, cytoskeleton, and nucleus. Immunoblotting of osteoarthritis cartilage were performed using polyclonal anti-clock antibody. The biological rhythm of mRNA of clock and clock-related genes was analyzed by real-time polymerase chain reaction (PCR). Clock gene was confirmed by real-time PCR to validate the microarray data. Other clock-related genes such as Per1 and Per2 were also expressed in chondrocytes, exhibiting biological rhythm after serum shock. The large-scale DNA microarray indicated that clock and other genes functionally associated with mechanical stress play an essential role in regulating the biological rhythm of chondrocyte metabolism in osteoarthritis cartilage.

A Clock Shock: Mouse CLOCK Is Not Required for Circadian Oscillator Function

The circadian clock mechanism in the mouse is composed of interlocking transcriptional feedback loops. Two transcription factors, CLOCK and BMAL1, are believed to be essential components of the circadian clock. We have used the Cre-LoxP system to generate whole-animal knockouts of CLOCK and evaluated the resultant circadian phenotypes. Surprisingly, CLOCK-deficient mice continue to express robust circadian rhythms in locomotor activity, although they do have altered responses to light. At the molecular and biochemical levels, clock gene mRNA and protein levels in both the master clock in the suprachiasmatic nuclei and a peripheral clock in the liver show alterations in the CLOCK-deficient animals, although the molecular feedback loops continue to function. Our data challenge a central feature of the current mammalian circadian clock model regarding the necessity of CLOCK:BMAL1 heterodimers for clock function.

Molecular Analysis of Clock Gene Expression in the Avian Brain

Birds are equipped with a complex circadian pacemaking system that regulates the rhythmicity of physiology and behavior. As with all organisms, transcriptional and translational feedback loops of clock genes represent the basic molecular mechanism of rhythm generation in birds. To investigate avian clock gene expression, partial cDNA sequences of six mammalian clock gene homologs (Bmal1, Clock, Per2, Per3, Cry1, and Cry2) and a novel avian cryptochrome gene (Cry4) were cloned from the house sparrow, a model system in circadian research. Expression patterns were analyzed by semi‐quantitative RT‐PCR and RNase protection assays using total RNA extracted from adult male house sparrow brains. With the exception of Cry4, pronounced rhythmic mRNA expression of all the clock genes analyzed was encountered, with mRNA levels varying considerably between the various genes. Although some basic features of the molecular circadian feedback loop appear to be similar between mammals and birds, the precise phase relationships of the clock gene mRNA rhythms relative to each other and to the light zeitgeber differ significantly between the house sparrow and mammals. Our results point to the existence of differences in the organization of avian and mammalian circadian clock mechanisms.

Ontogenesis of photoperiodic entrainment of the molecular core clockwork in the rat suprachiasmatic nucleus

The molecular mechanism underlying a generation of circadian rhythmicity within the suprachiasmatic nucleus (SCN) is based on interactive negative and positive feedback loops that drive the rhythmic transcription of clock genes and translation of their protein products. In adults, the molecular mechanism is affected by seasonal changes in day length, i.e., photoperiod. The photoperiod modulates phase, waveform, and amplitude of the rhythmic clock genes expression as well as the phase relationship between their profiles. To ascertain when and how the photoperiod affects the circadian core clock mechanism during ontogenesis, the rhythmic expression of clock genes, namely of Per1, Per2, Cry1 and Bmal1 was determined in 3-, 10- and 20-day-old rat pups maintained under either a long photoperiod with 16 h of light and 8 h of darkness per day (LD 16:8) or under a short, LD 8:16 photoperiod. The daily profiles in the level of clock genes mRNA were studied in constant darkness. The photoperiod affected the profile of Per1 and Per2 mRNA in 20- and 10- but not yet in 3-day-old pups. Expression of Cry1 was affected only in 20-day-old pups, whereas expression of Bmal1 was not yet affected even in 20-day-old rats. The results demonstrate no effect of the photoperiod on 3-day-old pups, only partial entrainment of the molecular core clockwork in 10-day-old pups and a more mature, though not yet fully complete, entrainment in 20-day-old pups as compared with adult animals. The developmental interval when the photoperiod begins to entrain the core clock mechanism completely might thus occur around the time of weaning.

Rapid damping of food-entrained circadian rhythm of clock gene expression in clock-defective peripheral tissues under fasting conditions

Restricted feeding-induced free-running oscillation of clock genes in the liver was studied in homozygous Clock-mutant ( Clock/ Clock) mice. Similar to wild-type mice, Clock/ Clock mice showed robust food-anticipatory behavioral activity in accordance with a restricted feeding schedule. Also, the peak of all clock gene mRNAs tested was phase-advanced in the liver of Clock/ Clock mice as well as wild-type mice, although the amplitude of clock gene expression was low in Clock/ Clock mice. The food-anticipatory behavioral rhythm in Clock/ Clock mice maintained a period similar to wild-type mice during 2-day fasting after the cessation of restricted feeding. However, during the fasting days after temporal feeding cues were removed, the oscillation of clock genes in the liver and heart, excluding the suprachiasmatic nuclei, appeared to result in arrhythmicity in Clock/ Clock mice. Thus, although the CLOCK-based molecular mechanism is not required for the expression of food-anticipatory activity, intact CLOCK protein might be involved in sustaining several cycles of peripheral circadian oscillations after restricted feeding-induced resetting.

Daily Expression Patterns of Cycle and Clock Genes in the Head of the Silkworm, Bombyx Mori

ABSTRACTClock and cycle (Bmall) genes encode the basic helix-loop-helix-PAS (Period-Aryl-hydrocarbon receptor-Singleminded) proteins that function as transcription factors regulating the transcription/translation-based autoregulatory feedback in the circadian clock of many organisms. In the present study we examined the temporal pattern in the expression of mRNA of Clock (BmClk) and Cycle (BmCyc) genes in the head of Bombyx mori by Northern blot analysis. BmClk gene was co-expressed in the same tissues as those where BmCyc was expressed. In a daily light-dark cycle BmCyc did not show significant difference in expression. Surprisingly unlike the case of Drosophila melanogaster, the levels of BmClk transcript exhibited no prominent circadian oscillation in the moth head. This casts a doubt that the structure and regulatory mechanism of circadian clock may not be uniform among organisms.