Lunar cycle-associated physiology has been found in a wide variety of organisms. Studies suggest the presence of a circalunar clock in some animals, but the location of the lunar clock is unclear. We previously found lunar-associated expression of transcripts for Cryptochrome3 gene (SgCry3) in the brain of a lunar phase-responsive fish, the Goldlined spinefoot (Siganus guttatus). Then we proposed a photoperiodic model for the lunar phase response, in which SgCry3 might function as a phase-specific light response gene and/or an oscillatory factor in unidentified circalunar clock. In this study, we have developed an anti-SgCRY3 antibody to identify SgCRY3-immunoreactive cells in the brain. We found immunoreactions in the subependymal cells located in the mediobasal region of the diencephalon, a crucial site for photoperiodic seasonal responses in birds. For further assessment of the lunar-responding mechanism and the circalunar clock, we investigated mRNA levels of Cry3 as well as those of the other clock(-related) genes, Period (Per2 and Per4), in S. guttatus reared under nocturnal moonlight interruption or natural conditions. Not only SgCry3 but SgPer4 mRNA levels showed lunar phase-dependent variations in the diencephalon without depending on light condition during the night. These results suggest that the expressions of SgCry3 and SgPer4 are not directly regulated by moonlight stimulation but endogenously mediated in the brain, and implicate that circadian clock(-related) genes may be involved in the circalunar clock locating within the mediobasal region of the diencephalon.

CRY proteins can be classified into several groups based on their phylogenetic relationships, and they function as a photoreceptor, a photolyase, and/or a transcriptional repressor of the circadian clock. In order to elucidate the expression profile and functional diversity of CRYs in vertebrates, we focused on XtCRY4, a member of the uncharacterized cryptochrome family CRY4 in Xenopus tropicalis. XtCRY4 cDNA was isolated by RT-PCR, and a phylogenetic analysis of deduced sequence of XtCRY4 suggested that the vertebrate Cry4 genes evolved at much higher evolutionary rates than mammalian-type Cry genes, such as the CRY1 and CRY2 circadian clock molecules. A transcriptional assay was performed to examine the transcriptional regulatory function as circadian repressor, and XtCRY4 had marginal effects on the transactivation of XtCLOCK/XtBMAL1 via E-box element. In situ hybridization and quantitative RT-PCR was performed to detect mRNA expression in native tissues. Quantitative RT-PCR revealed that XtCry4 mRNA was highly transcribed in the ovary. In situ hybridization showed the presence of XtCry4 transcripts in the oocytes, testis, renal tubules, the visual photoreceptors, and the retinal ganglion cells. A specific antiserum to XtCRY4 was developed to detect endogeneous expression of XtCRY4 protein in the ovary. The expression level was estimated by immunoblot analysis, and this is the first detection and estimation of endogenous expression of CRY protein in the ovary. These results suggest that X. tropicalis ovary may respond to blue-light by using XtCRY4.

Animals have several classes of cryptochromes (CRYs), some of which function as core elements of circadian clockwork, circadian photoreceptors, and/or light-dependent magnetoreceptors. In addition to the circadian clock genes Cry1 and Cry2, nonmammalian vertebrates have the Cry4 gene, the molecular function of which remains unknown. Here we analyzed chicken CRY4 (cCRY4) expression in the retina with in situ hybridization and found that cCRY4 was likely transcribed in the visual pigment cells, cells in the inner nuclear layer, and retinal ganglion cells. We further developed several monoclonal antibodies to the carboxyl-terminal extension of cCRY4 and localized cCRY4 protein with immunohistochemistry. Consistent with the results of in situ hybridization, cCRY4 immunoreactivity was found in visual pigment cells and cells located at the inner nuclear layer and the retinal ganglion cell layer. Among the antibodies, one termed C1-mAb had its epitope within the carboxyl-terminal 14-amino acid sequence (QLTRDDADDPMEMK) and associated with cCRY4 in the retinal soluble fraction more strongly in the dark than under blue light conditions. Immunoprecipitation experiments under various light conditions indicated that cCRY4 from the immunocomplex formed in the dark dissociated from C1-mAb during blue light illumination as weak as 25 mu W/cm(2) and that the release occurred with not only blue but also near UV light. These results suggest that cCRY4 reversibly changes its structure within the carboxyl-terminal region in a light-dependent manner and operates as a photoreceptor or magnetoreceptor with short wavelength sensitivity in the retina.

A variety of animals use Earth's magnetic field as a reference for their orientation behaviour. Although distinctive magnetoreception mechanisms have been postulated for many migrating or homing animals, the molecular mechanisms are still undefined. In this study, we found that zebrafish, a model organism suitable for genetic manipulation, responded to a magnetic field as weak as the geomagnetic field. Without any training, zebrafish were individually released into a circular arena that was placed in an artificial geomagnetic field, and their preferred magnetic directions were recorded. Individuals from five out of the seven zebrafish groups studied, groups mostly comprised of the offspring of predetermined pairs, showed bidirectional orientation with group-specific preferences regardless of close kinships. The preferred directions did not seem to depend on gender, age or surrounding environmental factors, implying that directional preference was genetically defined. The present findings may facilitate future study on the molecular mechanisms underlying magnetoreception.

Lunar cycle-associated physiology has been found in a wide variety of organisms. Recent study has revealed that mRNA levels of Cryptochrome (Cry), one of the circadian clock genes, were significantly higher on a full moon night than on a new moon night in coral, implying the involvement of a photoreception system in the lunar-synchronized spawning. To better establish the generalities surrounding such a mechanism and explore the underlying molecular mechanism, we focused on the relationship between lunar phase, Cry gene expression, and the spawning behavior in a lunar-synchronized spawner, the goldlined spinefoot (Siganus guttatus), and we identified two kinds of Cry genes in this animal. Their mRNA levels showed lunar cycle-dependent expression in the medial part of the brain (mesencephalon and diencephalon) peaking at the first quarter moon. Since this lunar phase coincided with the reproductive phase of the goldlined spinefoot, Cry gene expression was considered a state variable in the lunar phase recognition system. Based on the expression profiles of SgCrys together with the moonlight's pattern of timing and duration during its nightly lunar cycle, we have further speculated on a model of lunar phase recognition for reproductive control in the goldlined spinefoot, which integrates both moonlight and circadian signals in a manner similar to photoperiodic response.

Cryptochromes (CRYs) are flavoproteins sharing high homology with photolyases. Some of them have function(s) including transcription regulation in the circadian clock oscillation, blue-light photoreception for resetting the clock phase, and light-dependent magnetoreception. Vertebrates retain multiple sets of CRY or CRY-related genes, but their functions are yet unclear especially in the lower vertebrates. Although CRYs and the other circadian clock components have been extensively studied in the higher vertebrates such as mice, only a few model species have been studied in the lower vertebrates. In this study, we identified two CRYs, XtCRY1 and XtCRY2 in Xenopus tropicalis, an excellent experimental model species. Examination of tissue specificity of their mRNA expression by real-time PCR analysis revealed that both the XtCRYs showed extremely high mRNA expression levels in the ovary. The mRNA levels in the ovary were about 28-fold (XtCry1) and 48-fold (XtCry2) higher than levels in the next abundant tissues, the retina and kidney, respectively. For the functional analysis of the XtCRYs, we cloned circadian positive regulator XtCLOCK and XtBMAL1, and found circadian enhancer E-box in the upstream of XtPer1 gene. XtCLOCK and XtBMAL1 exhibited strong transactivation from the XtPer1 E-box element, and both the XtCRYs inhibited the XtCLOCK: XtBMAL1-mediated transactivation, thereby suggesting this element to drive the circadian transcription. These results revealed a conserved main feedback loop in the X. tropicalis circadian clockwork and imply a possible physiological importance of CRYs in the ovarian functions such as synthesis of steroid hormones and/or control of estrus cycles via the transcription regulation.

The circadian clock controls daily rhythms in many physiologic processes, and the clock oscillation is regulated by external time cues such as light, temperature, and feeding. In mammals, the transcriptional regulation of clock genes underlies the clock oscillatory mechanism, which is operative even in cultured fibroblasts. We previously demonstrated that glucose treatment of rat-1 fibroblasts evokes circadian expression of clock genes with a rapid induction of Tieg1 transcript encoding a transcriptional repressor. Here, we found diurnal variation of both Tieg1 mRNA and nuclear TIEG1 protein levels in the mouse liver with their peaks at day/night transition and midnight, respectively. In vitro experiments showed that TIEG1 bound to Bmal1 gene promoter and repressed its transcriptional activity through two juxtaposed GC boxes near the transcription initiation site. The GC box/TIEG1-mediated repression of Bmal1 promoter was additive to RORE-dependent repression by REV-ERB alpha, a well-known repressor of Bmal1 gene. In cell-based real-time assay, siRNA-mediated knock-down of TIEG1 caused period shortening of cellular bioluminescence rhythms driven by Bmal1-luciferase and Per2-luciferase reporters. These findings highlight an active role of TIEG1 in the normal clock oscillation and GC box-mediated regulation of Bmal1 transcription.

In the molecular oscillatory mechanism governing circadian rhythms, positive regulators, including CLOCK and BMAL1, transactivate Per and Cry genes through E-box elements, and translated PER and CRY proteins negatively regulate their own transactivation. Like BMAL1, its paralog BMAL2 dimerizes with CLOCK to activate the E-box-dependent transcription, but the role of BMAL2 in the circadian clockwork is still elusive. Here we characterized BMAL2 function in NIH3T3 cells and found that the cellular rhythms monitored by Bmal1 promoter-driven bioluminescence signals were blunted by RNA interference-mediated suppression of Bmal2 as well as that of Bmal1. Transcription assays with a 2.1-kb mPer1 promoter revealed that CRY2 inhibited the transactivation mediated by BMAL1-CLOCK more strongly than that by BMAL2-CLOCK. In contrast, PER2 showed a stronger inhibitory effect on BMAL2-CLOCK than on BMAL1-CLOCK. The molecular link between BMAL2 and PER2 was further strengthened by the fact that PER2 exhibited a greater affinity for BMAL2 than for BMAL1 in co-immunoprecipitation experiments. These results indicate a functional partnership between BMAL2 and PER2 and reemphasize the negative role of PER2 in the circadian transcription. As a broad spectrum function, BMAL2-CLOCK activated transcription from a variety of SV40-driven reporters harboring various E/E'-box-containing sequences present in the upstream regions of clock and clock-controlled genes. Importantly, the efficiencies of BMAL2-CLOCK-mediated transactivation relative to that achieved by BMAL1-CLOCK were dependent heavily on the E-box-containing sequences, supporting distinguishable roles of the two BMALs. Collectively, it is strongly suggested that BMAL2 plays an active role in the circadian transcription.

In mammalian circadian clockwork, the CLOCK-BMAL1 heterodimer activates E-box-dependent transcription, while its activity is suppressed by circadian binding with negative regulators, such as CRYs. Here, we found that the CLOCK protein is kept mostly in the phosphorylated form throughout the day and is partly hyperphosphorylated in the suppression phase of E-box-dependent transcription in the mouse liver and NIH 3T3 cells. Coexpression of CRY2 in NIH 3T3 cells inhibited the phosphorylation of CLOCK, whereas CIPC coexpression markedly stimulated phosphorylation, indicating that CLOCK phosphorylation is regulated by a combination of the negative regulators in the suppression phase. CLOCK-BMAL1 purified from the mouse liver was subjected to tandem mass spectrometry analysis, which identified Ser38, Ser42, and Ser427 as in vivo phosphorylation sites of CLOCK. Ser38Asp and Ser42Asp mutations of CLOCK additively and markedly weakened the transactivation activity of CLOCK-BMAL1, with downregulation of the nuclear amount of CLOCK and the DNA-binding activity. On the other hand, CLOCK Delta 19, lacking the CIPC-binding domain, was far less phosphorylated and much more stabilized than wild-type CLOCK in vivo. Calyculin A treatment of cultured NIH 3T3 cells promoted CLOCK phosphorylation and facilitated its proteasomal degradation. Together, these results show that CLOCK phosphorylation contributes to the suppression of CLOCK-BMAL1-mediated transactivation through dual regulation: inhibition of CLOCK activity and promotion of its degradation.

To better understand dermal response to visible light, we used DNA microarray analysis to search genes induced by blue or near-UV light in normal human dermal fibroblasts. Of about 12800 transcripts analyzed, near- UV light most prominently upregulated the transcript level of Mic-1, a gene encoding a TGF-beta superfamily protein. Quantitative RT-PCR and immunoblot analyses revealed that mRNA and protein levels of Mic-1 were upregulated by both short-wavelength light but not by green or red light. These results suggest that the human dermis is a site for macrophage inhibitory cytokine-1 (MIC-1) production and that visible light activates a dermal transcription cascade. Considering the role of MIC-1 in immune regulation and appetite control, photic MIC-1 regulation is of physiological importance. (C) 2009 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

It is now known that circadian clocks are localized not only in the central pacemaker but also in peripheral organs. An example of a clock-dependent peripheral organ is the ovary of domestic poultry in which ovulation is induced by the positive feedback action of ovarian progesterone on the neuroendocrine system to generate a preovulatory release of LH during a daily 6-10 h "open period" of the ovulatory cycle. It has been assumed previously that the timing of ovulation in poultry is controlled solely by a clock-dependent mechanism within the neuroendocrine system. Here, we question this assumption by demonstrating the expression of the clock genes, Per2 (Period 2) and Per3, Clock, and Bmal1 (brain and muscle Arnt-like protein 1), in preovulatory follicles in laying quail. Diurnal changes in Per2 and Per3 expression were seen in the largest preovulatory follicle (F1) but not in smaller follicles. We sought to identify clock-driven genes in preovulatory follicles focusing on those involved in the synthesis of progesterone. One such gene was identified, encoding steroidogenic acute regulatory protein (StAR), which showed 24-h changes in expression in the F1 follicle coinciding with those of Per2. Evidence that StAR gene expression is clock driven was obtained by showing that its 5' flanking region contains E-box enhancers that bind to CLOCK/BMAL1 heterodimers to activate gene transcription. We also showed that LH administration increased the promoter activity of chicken StAR. We therefore suggest that the timing of ovulation in poultry involves an LH-responsive F1 follicular clock that is involved in the timing of the preovulatory release of progesterone.

Retinal cone cells exhibit distinctive photoresponse with a more restrained sensitivity to light and a more rapid shutoff kinetics than those of rods. To understand the molecular basis for these characteristics of cone responses, we focused on the opsin deactivation process initiated by G protein-coupled receptor kinase (GRK) 1 and GRK7 in the zebrafish, an animal model suitable for studies on retinal physiology and biochemistry. Screening of the ocular cDNAs identified two homologs for each of GRK1 (1A and 1B) and GRK7 (7-1 and 7-2), and they were classified into three GRK subfamilies, 1 A, 1B and 7 by phylogenetic analysis. In situ hybridization and immunohistochemical studies localized both GRK1B and GRK7-1 in the cone outer segments and GRK1A in the rod outer segments. The opsin/GRKs molar ratio was estimated to be 569 in the rod and 153 in the cone. The recombinant GRKs phosphorylated light-activated rhodopsin, and the V-max value of the major cone subtype, GRK7-1, was 32-fold higher than that of the rod kinase, GRK1A. The reinforced activity of the cone kinase should provide a strengthened shutoff mechanism of the light-signaling in the cone and contribute to the characteristics of the cone responses by reducing signal amplification efficiency.

In non-mammalian vertebrates, the pineal gland contains an endogenous circadian oscillator and serves as a photosensitive neuroendocrinal organ. To better understand the pineal phototransduction mechanism, we focused on the chicken putative blue-light photoreceptive molecule, Cryptochrome4 (cCRY4). Here we report the molecular cloning of pineal cCry4 cDNA, the in vivo expression of cCry4 mRNA, and the detection of cCRY4 protein. cCry4 is transcribed in a wide variety of chick tissues out of which the pineal gland and retina contain high levels of cCry4 mRNA. In the pineal gland, under 12 h light : 12 h dark cycles, the levels of both cCry4 mRNA and cCRY4 protein showed diurnal changes, and in cultured chick pineal cells, the cCry4 mRNA level was not only up-regulated by light but also controlled by circadian signals. Immunoblot analysis with a cCRY4-specific antibody detected cCRY4 in a soluble fraction of the pineal lysate. Immunocytochemistry revealed that cCRY4 was expressed in many parenchymal cells and a limited number of stromal cells. These cCRY4 features strikingly contrast with those of the chick pineal photoreceptor pinopsin, suggesting a possible temporal and/or spatial duplicity of the pineal photoreceptive system, the opsin- and CRY-based mechanisms.

The circadian clock is an autonomous biological clock that is entrainable to environmental 24-h cycles by receiving time cues such as light. Generally, light given at early and late subjective night, respectively, delays and advances the phase of the circadian oscillator. We previously searched for the chicken pineal genes that are induced by light in a phase-dependent manner. The present study undertook cDNA cloning and characterization of a gene whose expression was remarkably up-regulated by light at late subjective night. The mRNA level of this gene exhibited robust diurnal change in the pineal gland, with a peak in the early (subjective) day under light-dark cycles and constant dark condition, and hence it was designated Lcg (Light-inducible and Clock-controlled Gene). Chicken Lcg encodes a coiled-coil protein composed of 560 amino acid residues. Among chicken tissues, the pineal gland and the retina exhibited relatively high expression levels of LCG. LCG was colocalized with gamma-tubulin, a centrosomal protein, when expressed in COS7 cells, and LCG is the first example of a clock-related molecule being accumulated at the centrosome. Coimmunoprecipitation of LCG with gamma-tubulin in the chicken pineal lysate suggests a link between the circadian oscillator and the centrosomal function.

Light-dependent transcriptional regulation of clock genes is a crucial step in the entrainment of the circadian clock [1]. E4bp4 is a light-inducible gene in the chick pineal gland [2], and it encodes a bZIP protein that represses transcription of cPer2, a chick pineal clock gene [2,3]. Here, we demonstrate that prolonged light period-dependent accumulation of E4BP4 protein is temporally coordinated with a delay of the rising phase of cPer2 in the morning. E4BP4 was phosphorylated progressively and then disappeared in parallel with induced cPer2 expression. Characterization of E4BP4 revealed Ser182, a phosphoacceptor site located at the amino-terminal border of the Ser/Thr cluster, which forms the phosphorylation motifs for casein kinase 1epsilon (CK1epsilon). CK1epsilon physically associated with E4BP4 and phosphorylated it. CK1epsilon-catalyzed phosphorylation of E4BP4 resulted in proteasomal proteolysis-dependent decrease of E4BP4 levels, while E4BP4 nuclear accumulation was attenuated by CK1epsilon in a kinase activity-independent manner. CK1epsilon-mediated posttranslational regulation was accompanied by reduction of the transcriptional repression executed by E4BP4. These results not only demonstrate a phosphorylation-dependent regulatory mechanism for E4BP4 function but also highlight the role of CK1epsilon as a negative regulator for E4BP4-mediated repression of cPer2.

In mammals, peripheral circadian clocks are present in most tissues, but little is known about how these clocks are synchronized with the ambient 24-h cycles. By using rat-1 fibroblasts, a model cell system of the peripheral clock, we found that an exchange of the culture medium triggered circadian gene expression that was preceded by slow down-regulation of Per1 and Per2 mRNA levels. This profile contrasts to the immediate up-regulation of these genes often observed for clock resetting. The screening of factor(s) responsible for the down-regulation revealed glucose as a key component triggering the circadian rhythm. The requirement of both glucose metabolism and RNA/protein synthesis for the down-regulation suggests the involvement of gene(s) immediately up-regulated by glucose metabolism. An analysis with high density oligonucleotide microarrays identified >100 glucose-regulated genes. We found among others immediately up-regulated genes encoding transcriptional regulators TIEG1, VDUP1, and HES1, in addition to cooperatively regulated genes that are associated with cholesterol biosynthesis and cell cycle. The immediate up-regulation of Tieg1 and Vdup1 expression was dependent on glucose metabolism but not on protein synthesis, suggesting that the transcriptional regulators mediate the glucose-induced down-regulation of Per1 and Per2 expression. These results illustrate a novel mode of peripheral clock resetting by external glucose, a major food metabolite.

Light is a major environmental signal for entrainment of the circadian clock, but little is known about the intracellular phototransduction pathway triggered by light activation of the photoreceptive molecule(s) responsible for the phase shift of the clock in vertebrates. The chicken pineal gland and retina contain the autonomous circadian oscillators together with the photic entrainment pathway, and hence they represent useful experimental models for the clock system. Here we show the expression of G(11)alpha, an alpha subunit of heterotrimeric G-protein, in both tissues by cDNA cloning, Northern blot, and Western blot analyses. G(11)alpha immunoreactivity was colocalized with pinopsin in the chicken pineal cells and also with rhodopsin in the outer segments of retinal photoreceptor cells, suggesting functional coupling of G(11)alpha with opsins in the clock-containing photosensitive tissues. The physical interaction was examined by coimmunoprecipitation experiments, the results of which provided evidence for light- and GTP-dependent coupling between rhodopsin and G(11)alpha. To examine whether activation of endogenous G(11) leads to a phase shift of the oscillator, G(q/11)-coupled m1-type muscarinic acetylcholine receptor (mAChR) was ectopically expressed in the cultured pineal cells. Subsequent treatment of the cells with carbamylcholine (CCh), an agonist of mAChR, induced phase-dependent phase shifts of the melatonin rhythm in a manner very similar to the effect of light. In contrast, CCh treatment induced no measurable effect on the rhythm of nontransfected (control) cells or cells expressing G(i/o)-coupled m2-type mAChR, indicating selectivity of the G-protein activation. Together, our results demonstrate the existence of a G(11)-mediated opsin-signaling pathway contributing to the photic entrainment of the circadian clock.

In vertebrates, a variety of light-stimulated genes are distributed in the retina, the pineal gland, and the suprachiasmatic nucleus, but a cis-element(s) responsible for the light-dependent transcriptional regulation is left unexplored. Focusing on the pinopsin gene, a light-stimulated gene in the chick pineal gland, we performed a transcriptional analysis in the primary culture of the chick pineal cells that were transiently transfected with a luciferase reporter gene fused with various lengths of the 5' upstream region of the pinopsin gene. Light-dependent enhancer activity was detectable in the construct with the upstream region between -1156 and +31. Introduction of mutations within the 18 bp sequence at positions -1103 to -1086 (TGGCACGTGGGGTTCCTC), including a CACGTG E-box sequence, elevated the transcriptional activity in the dark and thereby abrogated the light dependency, suggesting that the 18 bp sequence is essential for a reduction of the transcriptional activity in the dark. In an electrophoretic mobility-shift assay, we identified a pineal nuclear factor(s) capable of binding to the 18 bp element in a sequence-specific manner. When a 49 bp fragment (-1122 to -1074) including the 18 bp sequence was placed upstream of the simian virus 40 promoter, the transcriptional activity was dramatically suppressed regardless of light conditions in the chick pineal cells, and a more pronounced repression was observed in nonpineal/nonphotosensory LMH and NIH 3T3 cells. These results suggest that the 18 bp element in the pinopsin promoter constitutes the binding site of a ubiquitous factor that serves for the transcriptional repression that is required, although not sufficient, for the light-dependent expression of pinopsin gene in the chick pinealocytes.

The circadian clock is entrained to the diurnal alteration of environmental conditions such as light and temperature, but the molecular mechanism underlying the entrainment is not fully understood. In the present study, we employed a differential display-based screening for a set of genes that are induced by light in the chick pineal gland, a structure of the central clock entrainable to both light and temperature changes. We found that the level of the mRNA encoding chicken heat shock protein 90 alpha (cHSP90alpha) was rapidly elevated in the pineal gland within a 5-min exposure of chicks to light. Furthermore, the pineal cHsp90alpha mRNA was expressed rhythmically under both 12-hr light/12-hr dark (LD) cycles and constant dark (DD) conditions. The total amount of the pineal cHSP90a protein was, however, kept at nearly constant levels under LD cycles, and immunohistochemical analyses of the pineal cHSP90a showed invariable localization at the cytoplasm throughout the day. In vivo measurement of the chick pineal temperature demonstrated its light-dependent and time-of-day-dependent change, and the profile was very similar to that of the pineal cHsp90a mRNA level. These observations suggest that the in vivo temperature change regulates the expression of temperature-responsive genes including cHsp90a in the pineal gland. The temperature change may induce a phase-shift of the pineal clock, thereby facilitating its efficient entrainment to environmental LD cycles.

Vertebrate cryptochrome homologs (CRYs) are negative regulators for the transcription/translation-based autoregulatory feedback loop of the circadian clock. In this study we identified two Cry genes in the chicken, cCry1 and cCry2, which are expressed in the pineal gland. Messenger RNA levels of both cCry1 and cCry2 displayed circadian oscillation in cultured pineal cells under light/dark and constant darkness conditions. Noticeably, their mRNA levels during the light period were significantly higher than those in the dark, indicating light-dependent up-regulation of the two Cry genes mediated by photoreceptor(s) intrinsic to the chick pineal cells. These cCRYs inhibited E-box element-dependent cBMAL1/2-cCLOCK-induced transcription, suggesting that the chick pineal circadian oscillator is composed of molecules that are functionally similar to those of mammals but are subject to light-regulation distinct from the mammalian clockwork. (C) 2001 Elsevier Science Ireland Ltd. All rights reserved.

Background: In a transcription/translation-based autoregulatory feedback loop of vertebrate circadian clock systems, a BMAL1-CLOCK heterodimer is a positive regulator for the transcription of the negative element gene Per. The chicken pineal gland represents a photosensitive clock tissue, but the pineal clock genes constituting the oscillator loop have been less well characterized.
Results: We identified expression of the Per2, Bmal1, Bmal2 and Clock genes in the chicken pineal gland. Messenger RNA levels of these genes exhibited overt circadian rhythms in the pineal cells, both in vivo and in culture. In vitro functional analyses revealed the formation of cBMAL1-cCLOCK and cBMAL2-cCLOCK heteromers. Both of the cBMAL-cCLOCK heteromers activated E-box element-dependent transcription, which was negatively regulated by cPER2 in luciferase assays. Co-expression of cCLOCK, cBMAL1 and cBMAL2 co-operatively activated E-box element-dependent transcription, and a greater level of expression of cBMAL2 inhibited the activation. In the cultured pineal cells, an over-expression of either cBMAL1 or cBMAL2 disrupted the circadian rhythm of melatonin production.
Conclusion: The functional characterization of the chicken pineal clock molecules supports the key roles of BMAL1, BMAL2 and CLOCK which contribute to the E-box-dependent transcriptional regulation in the circadian clock system.

The chicken pineal gland contains the autonomous circadian oscillator together with the photic-input pathway. We searched for chicken pineal genes that are induced by light in a time-of-day-dependent manner, and isolated chicken homolog of bZIP transcription factor E4bp4 (cE4bp4) showing high similarity to vrille, one of the Drosophila clock genes. cE4bp4 was expressed rhythmically in the pineal gland with a peak at very early (subjective) night under both 12-h light/12-h dark cycle and constant dark conditions, and the phase was nearly opposite to the expression rhythm of cPer2, a chicken pineal clock gene. Luciferase reporter gene assays showed that cE4BP4 represses cPer2 promoter through a E4BP4-recognition sequence present in the 5 ' -flanking region, indicating that cE4BP4 can down-regulate the chick pineal cPer2 expression. In vivo light-perturbation studies showed that the prolongation of the light period to early subjective night maintained the high level expression of the pineal cE4bp4, and presumably as a consequence delayed the onset of the induction of the pineal cPer2 expression in the next morning. These light-dependent changes in the mRNA levels of the pineal cE46p4 and cPer2 were followed by a phase-delay of the subsequent cycles of cE4bp4/cPer2 expression, suggesting that cE4BP4 plays an important role in the phase-delaying process as a light-dependent suppressor of cPer2 gene.

Extraretinal photoreceptor cells have been found in the pineal complex and deep brain of a variety of non-mammalian vertebrates. Light signals received by these photoreceptor cells seem to be a potent regulator of diverse physiological responses. Here, the pineal complex and deep brain of the Japanese grass lizard, Takydromus tachydromoides, were immunohistochemically analyzed to localize the photoreceptive molecule (opsin) and the light signal-transducing G-protein (transducin). In addition to the pineal organ and parietal eye constituting the pineal complex, we unexpectedly found a parapineal organ, which is located just below the parietal eye and is morphologically similar to the pineal organ. Both organs had photoreceptor-like cells with outer segments immunostained by anti-rhodopsin and anti-pinopsin antibodies. Neither opsin- nor transducin-like immunoreactivities were detected in the parietal eye with all the antibodies tested in this study, although its morphology resembles that of the lateral eyes. In the deep brain region, rhodopsin-like immunoreactivities were observed in the posterior pallial commissure and median eminence. The cerebrospinal fluid-contacting neurons in the paraventricular organ were immunoreactive to an antibody against cc-subunit of cone transducin. In lizards, this is the first report showing (1) rhodopsin- and pinopsin-like immunoreactivities in the parapineal organ, (2) rhodopsin-like immunoreactivity in the deep brain, and (3) putative photoreceptive areas in the hypothalamus.

Brain-Muscle-Arnt-Like-protein 2 (BMAL2; Arnt4) (aryl hydrocarbon receptor nuclear translocator) is a recently identified basic Helix-Loop-Helix-Per-Arnt-Sim (bHLH-PAS) transcription factor, which contributes to a positive regulation of autoregulatory feedback loop in vertebrate circadian clock systems. In this study, we cloned cDNAs encoding mouse and rat BMAL2 (mBMAL2 and rBMAL2) from mouse midbrain and rat-1 fibroblast cells, respectively. A phylogenetic analysis strongly suggested that vertebrate Bmal1 and Bmal2 genes were generated by a single gene duplication of an ancestral Bmal gene, a vertebrate ortholog of dCyc gene, and that 'BMAL2's putatively termed so far are orthologous. Interestingly, BMAL2 proteins have diverged about 20-fold more rapidly than BMAL1 proteins after the duplication, suggesting an as-yet-unidentified function conserved in BMAL1 but not in BMAL2. mBmal2 mRNA was constitutively expressed throughout the day under light-dark cycle in the mouse hypothalamus containing suprachiasmatic nucleus, the site of the central circadian oscillator in mammals. (C) 2001 Elsevier Science Ireland Ltd. All rights reserved.

Pinopsin is a chicken pineal photoreceptive molecule with a possible role in photoentrainment of the circadian clock. Sequence comparison among members of the rhodopsin family has suggested that pinopsin might have properties more similar to cone visual pigments than to rhodopsin, but the lifetime of the physiologically active intermediate (meta II) of pinopsin is rather similar to that of metarhodopsin II, which is far more stable than meta II intermediates of cone visual pigments [Nakamura, A. et al., (1999) Biochemistry 38, 14738-14745]. In the present study, we investigated the amino acid residue(s) contributing to this unique property of pinopsin by using site-directed mutagenesis to pinopsin-specific structural features, (i) Ser171, (ii) Asn184, and (iii) the second extracellular loop two-amino acids shorter than that of cone visual pigments. The meta II stability of the 171/184 double mutant of pinopsin (S171R/N184D) is almost the same as that of wild-type pinopsin. In contrast, the meta II lifetime is markedly shortened (one third) by introduction of the third mutation (replacement of a six-amino acid stretch, 188-193, by the corresponding eight residues of chicken green-sensitive cone pigment) to the 171/184 double mutant of pinopsin. Consistently, meta II of the green-sensitive pigment mutant, in which the eight-amino acid stretch is inversely replaced by the corresponding six residues of pinopsin, is more stable than meta II of the wild-type pigment. These results strongly suggest that the specific sequence and/or the number of residues at amino acids 188-193 in pinopsin play an important role in the stabilization of the meta II intermediate.

Melatonin is secreted from the pineal gland in a circadian manner. It is well established that the synthesis of melatonin shows a diurnal rhythm reflecting a daily change in serotonin N-acetyltransferase (NAT) activity, and the overall secretion of melatonin requires a cellular release process, which is poorly understood. To investigate the possible involvement of Golgi-derived vesicles in the release, we examined the effect of brefeldin A (BFA), a reversible inhibitor of Golgi-mediated secretion, on melatonin secretion of cultured chick pineal cells. We show here that treatment with BFA completely disassembles the Golgi apparatus and reduces melatonin secretion. In more detailed time course experiments, however, the inhibition of melatonin secretion is only observed after the removal of BFA in parallel with the reassembly of the Golgi apparatus. This inhibition of melatonin secretion is not accompanied by accumulation of melatonin in the cells. These observations indicate that chick pineal melatonin is released independently of the Golgi-derived vesicles, and suggest inhibition of melatonin synthesis after the removal of BFA. By measuring the activities and mRNA levels of melatonin-synthesizing enzymes, we found that the removal of BFA specifically inhibits NAT activity at the protein level. On the other hand, BFA causes no detectable phase-shift of the chick pineal oscillator regulating the circadian rhythm of melatonin secretion. The results presented here suggest that the Golgi-mediated vesicular transport is involved in neither the melatonin release nor the time-keeping mechanism of the circadian oscillator, but rather contributes to the regulation of NAT activity.

Photoreceptive areas responsible for the regulation of photoperiodicity have been localized in the brain of some avian species.We found that immunoreactivities to rhodopsin and the a-subunit of rod-type transducin (Gt(1)alpha) were colocalized in cerebrospinal fluid (CSF) contacting neurons in the pigeon lateral septum, a possible site for photoreception. Furthermore, RT-PCR analyses showed specicific gene expression of both cGMP-phosphodiesterase beta -subunit and conetype cGMP-gated cation channel alpha -subunit in this region. These results suggest that several components in rod/cone photoreceptors compositely contribute to the deep encephalic phototransduction cascade. (C) 2000 Wiley-Liss, Inc.

The chicken pineal gland is a photosensitive neuroendocrine organ producing melatonin in circadian clock-regulated and light-sensitive manners. To understand the relationship between the photoreceptive molecule pinopsin and the light-dependent melatonin suppression that is sensitive to pertussis toxin treatment, we have searched for pertussis toxin-sensitive G protein alpha-subunits expressed in the chicken pineal gland, Here we report the cDNA cloning of the pineal transducin alpha-subunit (Gt alpha), which is highly homologous to human retinal rod cell-specific Gt(1)alpha. Concurrent cDNA cloning of chicken retinal Gt(1)alpha and Gt(2)alpha (rod and cone cell-specific alpha-subunits of transducin, respectively) revealed that the chicken pineal Gt alpha is identical to the retinal Gt(1)alpha. Double-immunostaining analysis of the chicken pineal sections localized Gt(1)alpha-immunoreactivity in the rudimentary outer segments of both follicular and parafollicular pinealocytes that were immunopositive to anti-pinopsin antibody. To examine whether pineal Gt(1)alpha is involved in the pineal phototransduction pathway, trypsin protection assay was applied for detecting the conversion of GDP-bound Gt(1)alpha into the guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S)-bound form in the pineal membrane homogenate, It was clearly demonstrated that the pineal Gt(1)alpha is activated in a light-dependent manner in the presence of GTP gamma S. These data together suggest strongly that pineal Gt(1)alpha mediates the phototransduction pathway triggered by pinopsin in the chicken pinealocytes.

Pinopsin is a photoreceptive molecule present in the outer segments of chicken pinealocytes. In this paper, the localization of a-subunits of G-proteins, rod transducin (Gt1) and Gq/11, was examined by immunoelectron microscopy to investigate whether these G-proteins colocalize with pinopsin in the outer segments. Ultrathin sections of the chicken pineal gland were double-immunolabeled with antibodies to pinopsin and either Gt1 alpha or Gq/11 alpha. As shown previously, the outer segments around the follicular lumen exhibited divergent morphology with ciliary, bulbous, or lamellate shapes, and most of them displayed pinopsin immunoreactivity. The majority (>90%) of pinopsin-immunopositive outer segments were labeled by anti-Gt1 alpha and/or anti-Gq/11 alpha antibodies. Application of double-immunolabeling to serial sections demonstrated that a large number of the pinopsin-immunopositive outer segments contained both Gt1 alpha and Gq/11 alpha immunoreactivities. These results suggest that Gt1 alpha and Gq/11 alpha are functionally coupled with light-activated pinopsin within a single outer segment.

The pineal complex, deep brain, and skin have been known to function as extraretinal photoreceptors in non-mammalian vertebrates. To see the diversity of localization of extraretinal photoreceptors in lower vertebrates having different habitats, we analyzed the opsin-like immunoreactivities in anuran amphibians, Xenopus laevis, Rana catesbeiana, Rana: nigromaculata, and Bufo japonicus. An antiserum (toad Rh-AS) was raised against rhodopsin purified from the retinas of Japanese toad, B. japonicus. In the retina of all the anurans examined, the outer segments of rods were immunopositive to toad Rh-AS. The outer segments of most pinealocytes were immunopositive in R. catesbeiana, R. nigromaculata, and B. japonicus. The outer segments of photoreceptor-like cells within the frontal organ of R. nigromaculata were immunostained. Interestingly, toad Rh-AS immunostained many secretory cells of mucous glands in the head skin of B. japonicus, implying the presence of a novel photoreceptive molecule. Within the hypothalamus, toad Rh-AS immunostained many cells in the magnocellular preoptic nucleus of R. catesbeiana and B. japonicus. Toad Rh-AS also labeled cerebrospinal fluid (CSF)-contacting cells in the anterior preoptic nucleus of R. nigromaculata and those adjacent to the lateral ventricle within the septum of R. catesbeiana. Thus the distribution patterns of the rhodopsin-like immunoreactivities among the anurans were highly diverged, and there was no relationship between the distribution patterns and their habitats. (C) 2000 Wiley-Liss, Inc.

A circadian pacemaker generates a rhythm with a period of similar to 24 hr even in the absence of environmental time cues. Several photosensitive neuronal tissues such as the retina and pineal gland contain the autonomous circadian pacemaker together with the photic-input pathway responsible for entrainment of the pacemaker to the daily light/dark cycle. We show here that, in constant darkness, chick pineal mitogen-activated protein kinase (MAPK) exhibited an in vivo circadian rhythm in tyrosine phosphorylation and in enzymatic activity with a peak during subjective night. Phosphorylated and hence activated MAPK was rapidly dephosphorylated after light illumination during the nighttime when light induces a phase-shift of the pacemaker. The circadian rhythmicity in MAPK phosphorylation was also observed in the cultured pineal gland, and importantly, MAPK kinase inhibitor treatment during subjective night not only shifted the time-of-peak of MAPK phosphorylation but also induced a remarkable phase-delay of the circadian pacemaker. These results indicate an important role of MAPK for time keeping in circadian clock systems.

Chicken pineal pinopsin is the first example of extra-retinal opsins, but little is known about its molecular properties as compared with retinal rod and cone opsins. For characterization of extraretinal photon signaling, we have developed an overexpression system providing a sufficient amount of purified pinopsin. The recombinant pinopsin, together with similarly prepared chicken rhodopsin and green-sensitive cone pigment, was subjected to photochemical and biochemical analyses by using low-temperature spectroscopy and the transducin activation assay. At liquid nitrogen temperature (-196 degrees C), we detected two kinds of photoproducts, bathopinopsin and isopinopsin, having their absorption maxima (lambda(max)) at 527 and similar to 440 nm, respectively, and we observed complete photoreversibility among pinopsin, bathopinopsin, and isopinopsin. A close parallel of the photoreversibility to the rhodopsin system strongly suggests that light absorbed by pinopsin triggers the initial event of cis-trans isomerization of the 11-cis-retinylidene chromophore. Upon warming, bathopinopsin decayed through a series of photobleaching intermediates: lumipinopsin (lambda(max) 461 nm), metapinopsin I (460 nm), metapinopsin II (385 nm), and metapinopsin III (460 nm). Biochemical and kinetic analyses showed that metapinopsin II is a physiologically important photoproduct activating transducin. Detailed kinetic analyses revealed that the formation of metapinopsin IT is as fast as that of a chicken cone pigment, green, but that the decay process of metapinopsin II is as slow as that of the rod pigment, rhodopsin. These results indicate that pinopsin is a new type of pigment with a chimeric nature between rod and cone visual pigments in terms of the thermal behaviors of the meta II intermediate. Such a long-lived active state of pinopsin may play a role in the pineal-specific phototransduction process.

The avian pineal organ contains several. types of photoreceptors with different photopigments: rhodopsin, iodopsin, and pinopsin. We have previously examined the differentiation of both rhodopsin-like and iodopsin-like immunoreactive cells during pineal development in quail embryos to determine the onset of synthesis of specific proteins and their cellular localization. In the present study, we have performed pinopsin immunohistochemistry on in-vivo developing and in-vitro cultured pineal organs of quail embryos. The results were compared with these obtained with rhodopsin and iodopsin immunohistochemistry. In the developing pineal organs, pinopsin immunoreactivity was detected at embryonic day 8, i.e. five days earlier than rhodopsin-like and iodopsin-like immunoreactivities. It was localized exclusively in the protrusions extending into the lumen throughout development, whereas rhodopsin-like and iodopsin-like immunoreactivities were usually found both in cell bodies and processes. These differences were also observed under two different types of culture conditions (dissociated cell culture and organ culture) indicating that, in the avian pineal organ, the expression pattern of the pinopsin gene is basically different from those of the other two pineal photopigments. The present study suggests that pineal cells have a mechanism for the polarized transport of pinopsin molecules.

Melatonin synthesis in the retina as well as in the pineal gland exhibits daily variations with higher levels during the dark phase of light-dark cycles. To analyze the molecular mechanism of melatonin synthesis in the retina, we have cloned, sequenced and characterized a putative cDNA for arylalkylamine N-acetyltransferase (AANAT; EC 2.3.1.87), a rate-limiting enzyme in melatonin production, from the retina of the rainbow trout (Oncorhynchus mykiss). The trout AANAT cDNA (1,585 bp) contains an open reading frame encoding 240 amino acid protein (predicted molecular weight, 27,420) that is 51-65% identical to avian and mammalian AANAT. The trout retinal AANAT protein contains motifs A and B that are conserved among the N-acetyltransferase superfamily and eight potential phosphorylation sites. Southern blot analysis demonstrated that the protein is expressed by a single copy gene. A single AANAT transcript (1.6 kb) was detected in the retina but not in the liver by Northern blot analysis. The levels of AANAT mRNA in the retina exhibited day-night changes with 3.3-fold increase at night. These results indicate that in the rainbow trout retina, the activity of AANAT and thus melatonin synthesis are regulated at least in part at the transcriptional level.

The phase of a circadian clock in the chicken pineal gland is reset by an environmental light signal, which is captured by the pineal photoreceptive molecule(s), Here we show that the mRNA level of pinopsin, a predominant photoreceptive molecule in the pineal gland, undergoes a diurnal fluctuation in chickens maintained on a light/dark cycle, The mRNA levels in the light were approximately six times higher than those in the dark. This fluctuation was not observed in constant darkness, where the mRNA levels remained low, Subsequent light exposure of chickens increased the amount of pinopsin mRNA regardless of the circadian time. Clearly, the expression of pinopsin gene is controlled by a light signal, independent of the circadian clock. In vitro experiments using cultured pineal glands isolated from the visual system also revealed the light-dependent increase in pinopsin mRNA level, indicating that the pineal photoreceptive molecule(s) is responsible for the induction. These results demonstrate the presence of a feedback loop through which the light signal captured by pinopsin stimulates the transcription of its own gene in the chicken pineal gland, In contrast, pinopsin protein remained at an almost constant level in chickens maintained under the same light/dark cycles. The protein level, however, decreased to similar to 50% of the light/dark level under constant darkness and subsequently increased upon exposure to light after the dark period. It is suggested that, under the light/dark cycles, the pinopsin protein level is kept constant by the light-dependent synthesis, which would compensate for a possible degradation of pinopsin in the daytime.

We detected rhodopsin gene expression in the pigeon lateral septum, a photosensitive deep brain region that is responsible for the photoperiodic gonadal response. The nucleotide sequence of the deep brain rhodopsin cDNA clone exactly matched that of the retinal one, indicating that a single rhodopsin gene is transcribed in the two tissues. Immunohistochemical analysis localized rhodopsin in the cerebrospinal fluid-contacting neurons, which have been assumed to be photoreceptive cells in the deep brain. Pigeon rhodopsin seems to play dual important roles in the visual and non-visual systems, the latter of which contributes to the photoperiodic response. (C) 1998 Federation of European Biochemical Societies.

We have isolated a cDNA clone encoding a deep brain photoreceptive molecule from the hypothalamic cDNA library of the toad, Bufo japonicus. The deduced amino acid sequence showed the highest similarity to that of pinopsin (75-76%) among vertebrate retinal opsins, indicating the expression of toad pinopsin in the deep brain. Antibodies raised against the C-terminal tail of toad pinopsin stained cell bodies and the knoblike structures of the cerebrospinal fluid-contacting neurons in the anterior preoptic nucleus. This region is known to play an important role in breeding behavior, suggesting that toad pinopsin acts as a photosensor for the photoperiodic gonadal response. (C) 1998 Federation of European Biochemical Societies.

Pinopsin is a blue-sensitive photoreceptive molecule possibly involved in photic entrainment of the circadian pacemaker in the chicken pineal gland. To characterize pinopsin as a circadian photoreceptor, antibodies were raised against the C-terminal portion of pinopsin. Its expected from the divergence of the amino acid sequence of this region, the resultant antibody cross-reacted with neither chicken rhodopsin nor red-sensitive cone pigment (chicken red). in Western blot analysis, the antibody stained a single band of 42-kDa protein in a detergent-extract of chicken pineal membranes, suggesting that pinopsin (calculated molecular weight, 38 187) might be glycosylated and/or palmitoylated. Immunocytochemical examination of pineal sections of the chicken and the pigeon with this antibody revealed strong positive images for most of the membrane structures in the lumen of the follicles. This antibody also stained string- and bulb-shaped structures of the chicken parafollicular cells, the morphology of which resembles those of retinal photoreceptor cells. In contrast to the predominant distribution of pinopsin, a monoclonal antibody specific for chicken red stained a smaller number of membrane structures in the lumen of chicken pineal follicles. These results strongly suggest that the chicken pined gland contains at least two types of photoreceptive molecules, pinopsin (major) and chicken red (minor). We show that the former molecule is localized in parafollicular pinealocytes and in the outer segments of pinealocytes that make contact with the follicular lumen. (C) 1997 Elsevier Science B.V.

Pinopsin is a photoreceptive molecule cloned from the chicken pineal organ. An antibody highly specific for pinopsin was applied in light- and electron-microscopic immunocytochemical studies of the pineal organ of 1 to 2-month-old chickens. Intense immunoreactivity was found in the follicular lumen at the light-microscopic level. In addition, small immunoreactive spherical or fibrous structures were diffusely distributed at the parafollicular aspect of the pineal organ. To identify immunoreactive elements precisely, we used pre-embedding immunoelectron microscopy. These studies revealed immunoreactive outer segments of pinealocytes arranged closely side by side in the follicular lumina. The thin initial portion of the outer segment arose from a basal body located in the inner segment. Immunoreactive pear-shaped outer segments occupied small lumina. Follicular lumina displayed immunonegative arrays of whorl-like lamellar membranes. Occasionally, these immunonegative structures were surrounded by immunoreactive concentric lamellar complexes. In the parafollicular pineal parenchyma, long slender cilium-like structures or enlarged cilia and concentric lamellar arrays showed intense immunoreactivity. All immunoreactive structures observed in this study were considered to represent outer segments of pinealocytes of the chicken pineal organ.

The avian pinealocytes have an intrinsic circadian clock function that controls rhythmic synthesis of melatonin, and an environmental light signal can reset the phase of the clock. In addition to the photoendocrine function, the melatonin synthesis of the pinealocytes is regulated by neural signals from sympathetic nerves, Thus the avian pinealocytes show diagnostic characters which seem to represent an evolutionary transition from photosensory cells of lower vertebrates to the neuroendocrinal cells of mammals. To understand the evolutionary background of the regulatory mechanism for the melatonin synthesis in this organ, we screened the chicken pineal cDNA library to find alpha-subunits of heterotrimeric G-proteins involved in the photic and neural regulations, In addition to the transducin-like alpha-subunit (G(t) alpha) supposed to mediate the photic pathway, we isolated cDNA clones encoding G(i2)alpha, G(i3)alpha, and G(o1)alpha and its splicing variant G(o2)alpha. The deduced amino acid sequence of each G alpha had a potential site for pertussis toxin-catalyzed ADP-ribosylation, As it is known that adrenergic receptor-mediated inhibition of melatonin synthesis is blocked by pertussis toxin, the G-proteins identified in the present study are likely to contribute to this neuroendocrine function of the chicken pineal cells.

We have determined the primary structure of a novel gamma subunit (gamma(12), previously designated gamma(S1)) of G protein purified from bovine spleen. The mature gamma(12) protein composed of 68 amino acids had acetylated serine at the N terminus and geranylgeranylated/carboxylmethylated cysteine at the C terminus. This was consistent with the C-terminal prenylation signal in the amino acid sequence, which was predicted from gamma(12) cDNA isolated from a bovine spleen cDNA library. Western blots with the specific antibody against gamma(12) showed that gamma(12) is present in all tissues examined. Among various gamma subunits (gamma(1), gamma(2), gamma(3), gamma(7), and gamma(12)), gamma(12) has a unique property to be phosphorylated by protein kinase C. The phosphorylated amino acid residue was Ser(1) (or Ser(2)). The phosphorylated beta gamma(12) associated with G(o) alpha more tightly than the unphosphorylated form. Exposure of Swiss 3T3 and aortic smooth muscle cells to phorbol 12-myristate 13-acetate and NaF induced phosphorylation of gamma(12). Stimulation of aortic smooth muscle cells with natural vasoactive agents such as angiotensin II and vasopressin also induced phosphorylation of gamma(12). The extent of phosphorylation of beta gamma(12) in vitro was suppressed by a complex formation with G(o) alpha, which was relieved by the addition of guanosine 5'-O-(3-thiotriphosphate) or aluminum fluoride. These results strongly suggest that gamma(12) is phosphorylated by protein kinase C during activation of receptor(s) and G protein(s) in living cells.

We purified two kinds of visual pigments, gecko green and gecko blue, from retinas of Tokay geckos (Gekko gekko) by two steps of column chromatography, and investigated their photobleaching processes by means of low temperature spectroscopy. Absorption maxima of gecko green and blue solubilized in a mixture of 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) and phosphatidylcholine were 522 and 465 nm, respectively, which are close to those observed in the photoreceptor cells. Low temperature spectroscopy identified six intermediates in the photobleaching process of gecko green; batho (lambda(max) = 569 nm), BL (gamma(max) = 519 nm), lumi (507 nm), meta I (similar to 486 nm), meta II (similar to 384 nm), and meta HI intermediates (similar to 500 nm). In contrast to the high similarity in amino acid sequence between gecko green and iodopsin [Kojima, D., et al. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 6841-6845], the batho-green did not revert thermally to original gecko green but converts to the next intermediate. The photobleaching process of gecko blue was investigated by low temperature spectroscopy, and three intermediates, meta I (gamma(max) = similar to 470 nm), meta II (gamma(max) similar to 370 nm) and meta III (gamma(max) similar to 475 nm), were identified. A comparative study on the thermal behavior of meta intermediates revealed that the thermal stability of meta II intermediate of both of the gecko visual pigments is lower than that of metarhodopsin II. The result supports the idea that both the gecko visual pigments are cone-type ones.

IN avian pinealocytes, an environmental light signal resets the phase of the endogenous circadian pacemaker that controls the rhythmic production of melatonin(1-6). Investigation of the pineal phototransduction pathway should therefore reveal the molecular mechanism of the biological clock. The presence of rhodopsin-like photoreceptive pigment(4,5,7-9), transducin-like immunoreaction(10), and cyclic GMP-dependent cation-channel activity(11) in the avian pinealocytes suggests that there is a similarity between retinal rod cells and pinealocytes in the phototransduction pathway. We have now cloned chicken pineal cDNA encoding the photoreceptive molecule, which is 43-48% identical in amino-acid sequence to vertebrate retinal opsins. Pineal opsin, produced by transfection of complementary DNA into cultured cells, was reconstituted with 11-cis-retinal, resulting in formation of a blue-sensitive pigment (lambda(max) approximate to 470 nm). In the fight of this functional evidence and because the gene is specifically expressed only in the pineal gland, we conclude that it is a pineal photosensor and name it pinopsin.

The relative photosensitivity and the molar extinction coefficient of a highly purified iodopsin (chicken red sensitive cone visual pigment) solubilized in a mixture of 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate and phosphatidylchotine (CHAPS-PC) were measured using bovine rhodopsin solubilized in 2% digitonin as a standard and compared with those of chicken and bovine rhodopsins. The photosensitivity obtained (1.08) was close to those of rhodopsins (chicken, 1.04; bovine, 0.99) in CHAPS-PC. The molar extinction coefficient of iodopsin (47 200) was 1.15-1.17 times higher than those of rhodopsins (chicken, 40500; bovine, 41200). The oscillator strength of iodopsin (0.60) calculated from the extinction coefficient was nearly identical to that of chicken rhodopsin (0.61), suggesting that the chromophore of iodopsin is similar in configuration to rhodopsin. In contrast, the difference in quantum yield between iodopsin (0.62) and chicken rhodopsin (0.70) suggests that the chromophore-opsin interaction after absorption of a photon by the chromophore may be different.

The Tokay gecko (Gekko gekko), a nocturnal lizard, has two kinds of visual pigments, P467 and P521. In spite of the pure-rod morphology of the photoreceptor cells, the biochemical properties of P521 and P467 resemble those of iodopsin (the chicken red-sensitive cone visual pigment) and rhodopsin, respectively. We have found that the amino acid sequence of P521 deduced from the cDNA was very similar to that of iodopsin. In addition, P467 has the highest homology with the chicken green-sensitive cone visual pigment, although it also has a relatively high homology with rhodopsins. These results give additional strength to the transmutation theory of Walls [Walls, G. L. (1934) Am. J. Ophthalmol. 17, 892-915], who proposed that the rod-shaped photoreceptor cells of lizards have been derived from ancestral cone-like photoreceptors. Apparently amino acid sequences of visual pigments are less changeable than the morphology of the photoreceptor cells in the course of evolution.

The chicken retina contains rhodopsin (a rod visual pigment) and four kinds of cone visual pigments. The primary structures of chicken red (iodopsin) and rhodopsin have been determined previously. Here we report isolation of three cDNA clones encoding additional pigments from a chicken retinal cDNA library. Based on the partial amino acid sequences of the purified chicken visual pigments together with their biochemical and spectral properties, we have identified these clones as encoding the chicken green, blue, and violet visual pigments. Chicken violet was very similar to human blue not only in absorption maximum (chicken violet, 415 nm; human blue, 419 nm) but also in amino acid sequence (80.6% identical). Interestingly, chicken green was more similar (71-75.1%) than any other known cone pigment (42.0-53.7%) to vertebrate rhodopsins. The fourth additional cone pigment, chicken blue, had relatively low similarity (39.3-54.6%) in amino acid sequence to those of the other vertebrate visual pigments. A phylogenetic tree of vertebrate visual pigments constructed on the basis of amino acid identity indicated that an ancestral visual pigment evolved first into four groups (groups L, S, M1, and M2), each of which includes one of the chicken cone pigments, and that group Rh including vertebrate rhodopsins diverged from group M2 later. Thus, it is suggested that the gene for scotopic vision (rhodopsin) has evolved out of that for photopic vision (cone pigments). The divergence of rhodopsin from cone pigments was accompanied by an increase in negative net charge of the pigment.

Chicken pinealocytes contain three major components of the circadian clock system: 1) a self-sustained oscillator, 2) a photic-input pathway to the oscillator, and 3) an overt output represented by the rhythmic production of melatonin. Even under cultured conditions of isolated pineal gland or dissociated pinealocytes, the input-oscillator-output functions are well maintained. Because of these experimental advantages, chicken pineal gland has been one of the best models for the study of the circadian clock system. Since the finding of a pineal-specific photoreceptive molecule, pinopsin, we have characterized the endogenous phototransduction pathway in the pinealocytes. On the other hand, despite the long history of chick pineal research, the molecular mechanism underlying the pineal clock oscillation has been largely unknown. Our recent characterization of the chick pineal clock genes strongly suggests that they constitute a transcription/translation-based autoregulatory feedback loop, which is very similar to that generating circadian rhythmicity in mammalian SCN. © 2001 Wiley-Liss, Inc.

By using antibodies raised against toad rhodopsin (toad Rh-AS), bovine rhodopsin (bovine Rh-AS), chicken pinopsin (P9), toad pinopsin (PinC), bovine transducin (anti-pTr alpha, anti-pTc alpha), we applied immunocytochemistry to the retina, brain and skin of anuran amphibians. Most of the outer segments of the pinealocytes in the pineal organs were immunoreactive to toad Rh-AS in Rana catesbeiana, Rana nigromaculata and Bufo japonicus. In the frontal organ of R. nigromaculata, the outer segments of photoreceptor-like cells were immunoreactive to toad Rh-AS. No immunoreactivities to toad Rh-AS were observed in the pineal nor frontal organ of Xenopus laevis. Some secretory cells in mucous glands of the head skin of B. japonicus were immunopositive to toad Rh-AS. Those antibodies described above recognized many round and spindle-like cells in the hypothalamic nuclei such as the magnocellular preoptic nucleus, anterior preoptic nucleus and suprachiasmatic nucleus in R. catesbeiana. Therefore, the localization of immunopositive sites and the intensity of immunoreactivity were different among species and showed wide diversity depending on antibodies used. Retinoids were detected in the head skin of B. japonicus and the hypothalamus of R. catesbeiana by HPLC analysis.

The chicken pineal gland has an endogenous circadian oscillator that controls the diurnal oscillation of N-acetyltransferase activity responsible for melatonin rhythm. It has been speculated that the chicken pineal cell contains a photoreceptive molecule that receives the environmental light signal and transmits the signal to the oscillator for resetting the phase. In spite of several lines of evidence suggesting the similarity between retinal and pineal photon-signal transducing proteins, the identity of the photoreceptive molecule had been an open question. In 1994, we isolated a pineal cDNA encoding a novel photoreceptive molecule and named it "pinopsin." The protein expressed in 293EBNA cells bound 11-cis-retinal to form a blue-sensitive pigment with an absorption maximum at about 470 nm. A putative G-protein interaction site of pinopsin shared a relatively high similarity in amino acid sequence to that of rhodopsin, implying that pinopsin functionally couples with transducin or transducin-like G-protein(s) in the pineal cells. We have cloned a cDNA for chicken pineal transducin α-subunit, and the deduced amino acid sequence contained a potential site to be ADP-ribosylated by pertussis toxin (PTX). Therefore, the transducin-mediated pathway could be blocked by PTX, though previous studies showed that treatment of the cultured chicken pineal cells with PTX had no effect on the light-induced phase-shift of the oscillator. Accordingly, it is unlikely that transducin mediates the light-input pathway to the oscillator, which may involve PTX-insensitive G-protein(s) or some unidentified component(s). The G-protein coupled receptor-mediated signaling processes regulating melatonin synthesis are discussed. Copyright © Munksgaard. 1997. All rights reserved.

Determination of the primary structures of six kinds of vertebrate visual pigments enabled us to classify them into four groups of cone-type pigments. The phylogenetic tree demonstrated that an ancestor of vertebrate visual pigments evolved into four kinds of cone-type pigments, from one of which rhodopsins diverged. Tetrachromatic color vision of chicken is discussed on the basis of both the absorption spectra of purified cone pigments and the filtering effect of colored oil-droplets. © 1995.

Periodical change in hydrostatic pressure (tidal cycle) is one of potent environmental factors in aquatic environments. It remains unknown how tidal spawners utilize tidal cycle for synchronizing reproductive events. The present study aimed to clarify the physiological mechanism occurring in the brain of the threespot wrasse, which is a typical tidal spawner in coral reefs. Using a microarray of tiger puffer fish with hydrostatic pressure, we identified tidal-related genes. Some of them were identified in the brain of threespot wrasse. When hydrostatic pressure was given to the fish, mRNA abundance of C-type natriuretic peptide (Cnp) in the diencephalon increased. This result suggested that some genes in the brain act as a transducer of tidal stimuli

We have developed a system to control the light-swichable gene expression by using photoreceptive molecules. In order to polish this system toward application as a novel biological tool, we further characterize CRY photoreception and interaction proteins in this study. We have detected the positive fluorescent signals on the plasma membrane of mammalian cultured cells. We found relatively high expression levels of chicken cryptochrome in a constitutive expression system using budding yeast. We also succeeded in the purification of the cryptochrome protein having the chromophore FAD, investigation of the spectral properties, and detection of in vitro structural changes induced by light

交付申請書に記載の研究実施計画に沿って研究を進め、以下の知見を得た。
(1)概日時計の発振メカニズムを解明するためには、光による時計の同調メカニズムを調べることが重要である。本研究ではまず、松果体に発現するG蛋白質の遺伝子クローニングを行い、複数のサブクラスのG蛋白質(Gt,Gi2,Gi3,Go1,Go2)の全塩基配列を決定した[Okano et al.,in press]。また、研究代表者らが発見したニワトリ松果体の光受容蛋白質(ピノプシン)を認識する抗体を作成し、松果体におけるピノプシンの局在を調べた[Hirunagi et al.,in press]。その結果、これまで光受容細胞と推定されてきた松果体の瀘胞形成細胞以外にも多数の光受容細胞が存在することを確認した。現在、クローニングしたG蛋白質に特異的な抗体を用いた免疫組織学的解析によって、それぞれのG蛋白質の松果体内での局在を調べている。ピノプシンと共役するG蛋白質を同定することによって、光受容から時計のリセットに至る光情報伝達経路が推定できると期待している。
(2)生理学的研究から、年周リズムと関連する光受容体(Deep Brain Photoreceptor)が脳深部において発現していると推定されてきた。この脳深部光受容体の分子レベルでの実体は全く不明である。そこで本研究では、RT-PCR法を用いて光受容体遺伝子の高感度検索を行った。その結果、ヒキガエルの脳深部のmRNAから、網膜の光受容体よりも松果体の光受容体ピノプシンと非常に類似した光受容体遺伝子を単離することに成功した。現在、その全塩基配列の決定を行っている。

交付申請書に記載の研究実施計画に沿って研究を進め、以下の知見を得た。
1.ニワトリ松果体における概日時計の光位相調節メカニズムを知るため、光受容蛋白質ピノプシンの情報下流を検索した。具体的には、トランスデューシン類似のG蛋白質がピノプシンと共役して光情報を変換している可能性が高いと考え、ヒヨコ松果体cDNAライブラリーより、3量体G蛋白質αサブユニットの遺伝子を検索した。その結果、GT1α(桿体型トランスデューシン)・Go1α・Go2α・Gi2α・Gi3αをコードする5種類のcDNAが単離できたが、いずれも百日咳毒素によるADPリボシル化部位をもつことがわかった。松果体概日時計の光位相シフトが百日咳毒素に非感受性であることを考え併せると、これらのG蛋白質が時計の光位相シフトを媒介する可能性は低い。そこでさらに、特異抗体を用いて他のG蛋白質を探索したところ、Gq/11αが松果体に豊富に存在することを見出した。この遺伝子をヒヨコ松果体cDNAライブラリーより単離したところ、百日咳毒素によるADPリボシル化部位をもたないことがわかった。脊椎動物の光受容蛋白質はトランスデューシンGtと共役するが、イカ・タコ・ショウジョウバエなど無脊椎動物のロドプシンはGqと共役して光情報を変換増幅することが知られている。ニワトリ松果体という光受容組織においてピノプシンがGtならびにGqという2つのG蛋白質と共役しているとすれば、光情報変換のシステムとしての進化を考える上で極めて興味深い現象といえる。
2.ある種の動物の脳深部には長日・短日を認識する光受容体が存在し、季節性繁殖などの生理現象を司ると考えられているが、その分子実体は全く不明であった。本研究において、光受容体遺伝子の高感度検索を行った結果、ヒキガエルの脳深部mRNAから光受容蛋白質をコードすると考えられる遺伝子を単離することに成功した。

交付申請書に記載の研究実施計画に沿って研究を進め、以下の知見を得た。
【1】網膜以外の光受容組織における光情報伝達分子の構造と機能の解析
研究代表者らが発見した松果体の光受容体ピノプシンの情報伝達経路を明らかにするため、ヒヨコ松果体cDNAライブラリーより、三量体G蛋白質αサブユニットの遺伝子を検索した。その結果、Gt1α(桿体型トランスデューシンα)・Go1α・Go2α・Gi2α・Gi3αをコードすると思われる遺伝子が単離できたが、Gt2α(錐体型トランスデューシンα)遺伝子は得られなかった。塩基配列から推定されるアミノ酸配列では、ヒヨコ松果体Gt1αはウシ錐体Gt2αよりも明らかにウシ桿体Gt1αに高い相同性を示した(それぞれ97%と82%)。ノザンブロット解析の結果、やはりGt2α遺伝子はヒヨコ松果体ではほとんど発現していないことが判明した。これまでに知られている松果体の光情報伝達分子群は、ピノプシンを含めて錐体型であったが、桿体型G蛋白質αサブユニットが松果体に存在するという今回の結果は、網膜と松果体における情報伝達分子の進化を考える上で極めて興味深い。
一方、ピノプシンに対する特異的抗体を作成し、ヒヨコ松果体切片を免疫染色した結果、濾胞内腔に強い陽性像がみられ、ピノプシンは松果体細胞の外節に局在すると考えられた。現在、上記の各Gαに対する特異的抗体を用いてピノプシンと細胞内局在が一致するGαを同定しようと試みている。
【2】脳内光受容分子の同定とその一次構造解明
鳥類・両生類の脳深部にはロドプシン様の光受容体が存在し、概日リズムや季節性の生殖応答に関与するのではないかと考えられている。この分子の実体を知るため、ハト外側中隔部およびカエル視交叉上核のcDNAライブラリーから光受容体遺伝子を検索しているが、候補遺伝子はまだ得られていない。

交付申請書に記載の研究実施計画に沿って研究を進め、以下の知見を得た。【1】松果体細胞内の時刻情報をinvivoで可視化するために、ニワトリ松果体細胞を光受容能と時計発振機能を保持したまま分散培養するための条件を決定した。次に、顕微鏡下において連続的に灌流培養しながら個々の松果体細胞を赤外カメラで観察するシステムを完成した。これと並行して、時刻検出のためのプローブ分子の選択・設計へ向け、ニワトリ松果体細胞における光情報伝達経路を解析した。その結果、松果体に存在する桿体型トランスデューシン(Gt_1α)およびG_<11>αの全一次構造を決定することができた。さらに、これら2種類のG蛋白質が松果体外節において光受容蛋白質ピノプシンと共存することを明らかにした。【2】脊椎動物の脳深部(外側中隔、視床下部)には、光周性にかかわる光受容体があると推定されるが、細胞さえ同定されていない。これらの細胞において、時刻や日照時間がどのような形で情報化されているかを探り、その情報をin vivoで経時的に検出したい。そこでまず、脳深部の光受容蛋白質としては初めて、ヒキガエルとハトの脳深部に存在する光受容蛋白質を同定し、その一次構造を明らかにした。ヒキガエル脳深部光受容蛋白質は、松果体特異的光受容蛋白質ピノプシンと高いアミノ酸一致度を示した。一方、ハト脳深部の光受容蛋白質は、網膜のロドプシンと全く同一の分子であった。さらに、これら2種類の光受容蛋白質を特異的に認識する抗体を作成し免疫組織化学的解析を行った結果、ヒキガエル視床下部とハト外側中隔の脳脊髄液接触ニューロンがそれぞれ染色された

交付申請書に記載の研究実施計画に沿って研究を進め、以下の知見を得た。【松果体の光情報伝達経路と時計発振系の解析】ニワトリ松果体Gαの全一次構造決定に続いて、松果体のG蛋白質が光受容蛋白質ピノプシンと共存すること、Gt_1αおよびG_<11>αが光刺激依存的に光受容蛋白質から解離することを明らかにした。これらのことから、Gt_1を介した既知の光情報伝達経路の他に、G_<11>αを介した全く新しい経路が存在することが強く示唆された。これと並行して、ヒヨコ松果体細胞を用いたディファレンシャルディスプレイ解析を行い、時刻依存的に転写される遺伝子を探索した。その結果、分子シャペロンの一種であるHSP90遺伝子の転写量が概日時計によって調節されていると考えられた。また、概日時計への光入力系にチロシンリン酸化蛋白質が関与している可能性を検討した。その結果、ヒヨコ松果体MAPキナーゼが光刺激の直後に脱リン酸化されること、さらにMAPキナーゼのチロシンリン酸化量および活性は恒暗条件下においても概日リズムを示すことがわかった。また、MEKの阻害剤(PD98059)を培養松果体に投与すると、概日時計の位相シフトが観察された。これらのことから、MAPキナーゼは概日時計を構成する分子であり、MAPキナーゼの活性化・不活性化のサイクルは、概日時計が駆動するために必要であることが明らかになった。【脳深部における光情報伝達経路】脳深部(外側中隔、視床下部)に存在する光受容体を特異的に認識する抗体を作製し、免疫組織化学的手法により脳深部光受容細胞の形態や細胞内における光受容分子の局在について検討した。その結果、脳脊髄液接触ニューロンのノブ状構造および細胞膜上に光受容蛋白質が集積していることがわかった

交付申請書に記載の研究実施計画に沿って研究を進め、以下の知見を得た。【概日時計細胞の時刻情報可視化】顕微鏡観察下において連続的に灌流培養しながらニワトリ松果体細胞・ラット視交叉上核細胞を分散培養するシステムを完成した。次に個々の細胞の時刻を蛍光強度として検出するため、転写量が日周変動するマウスper1遺伝子のプロモーター領域をクローニングした。この下流に短寿命型GFPの遺伝子(d2EGFP)を繋ぎレポーターコンストラクトを作製した。【時計や光応答に関連する遺伝子の単離】松果体細胞を用いたディファレンシャルディスプレイ解析を行い、時刻依存的に転写される遺伝子を探索した。その結果、分子シャペロンの一種であるHSP90遺伝子の転写量が日周変動することを見出した。【松果体・脳深部の光情報伝達経路と時計発振系の解析】ニワトリ松果体Gαの全一次構造決定に続いて、松果体のG蛋白質が光受容蛋白質ピノプシンと共存すること、Gt_1αおよびG_<11>αが光刺激依存的に光受容蛋白質から解離することを明らかにした。これらのことから、Gt_1を介した既知の光情報伝達経路の他に、G_<11>αを介した全く新しい経路が存在することが強く示唆された。これと並行して、概日時計への光入力系にチロシンリン酸化蛋白質が関与している可能性を検討した。その結果、松果体MAPキナーゼが光刺激の直後に脱リン酸化されること、さらにMAPキナーゼのチロシンリン酸化量および活性は恒暗条件下においても概日リズムを示すことがわかった。また、MEKの阻害剤(PD98059)を培養松果体に投与すると、概日時計の位相シフトが観察された。これらのことから、MAPキナーゼは概日時計を構成する分子であり、MAPキナーゼの活性化・不活性化のサイクルは、概日時計が駆動するために必要であることが明らかになった

交付申請書に記載の研究実施計画に沿って研究を遂行し、下記の成果を得た。1.光依存的転写調節に関わる遺伝子配列(光応答エレメント)を同定するため、光に応答するピノプシン遺伝子のプロモータの解析を行った。その結果、18塩基からなる光応答エレメントを同定することに成功した。また、ゲルシフト解析の結果、この光エレメントに作用する因子は、肝臓等の種々の臓器に発現する転写抑制因子であることがわかった。2.概日時計発振系におけるMAPキナーゼの生理的役割の解明を目指して、咋年度に続きMAPキナーゼの標的分子を探索した。In vitroでのキナーゼアッセイの結果、BMAL1に加え、クリプトクローム(CRY1およびCRY2)がMAPキナーゼによってリン酸化されることが判明した。転写アッセイに基づく機能解析の結果、MAPキナーゼによるリン酸化に伴ってクリプトクロームの転写抑制能が修飾されることが分かった。そこで、リン酸化部位を特異的に認識する抗体を作製し、このようなリン酸化が生体内でおこっているかどうかを調べた。その結果、培養細胞において強制発現させたCRY1およびCRY2の特定のアミノ酸残基がMAPキナーゼによってリン酸化されることが明らかとなった。3.エクソロドプシンプロモータは、ゼブラフィッシュにおいて松果体特異的な遺伝子発現を誘導する。このプロモータを利用し、概日時計の発振における時計遺伝子産物(BMAL1およびBMAL2)の機能を明らかにすべく、これらBMALのドミナントネガティブ体を松果体特異的に発現するトランスジェニックゼブラフィッシュを複数系統樹立した。現在、これら個体のメラトニンリズムの解析を進めている。これと並行して、エクソロドプシンプロモータにおいて、松果体特異的な遺伝子発現を誘導するエレメントを決定しPIPE(Pineal Expression Promoting Element)と命名した

交付申請書に記載の研究実施計画に基づき、松果体・脳深部における生物時計の光入力系についての研究を遂行し、下記の成果を得た。【1】ニワトリの松果体の概日時計発振系を構成する時計分子の同定を目指して、松果体cDNAライブラリーから時計遺伝子の単離を試みた。その結果、CLOCK,PER2,BMAL1をそれぞれコードする全長cDNAを単離することができた。これに加え、BMAL1の新規ホモログのcDNAも単離し、コードされる蛋白質をBMAL2と命名した。ノザン解析の結果、これら4種類の時計遺伝子のmRNA量は概日時計によってコントロールされていることが判明した。機能解析の結果、BMAL2はBMAL1と同様にCLOCKと2量体を形成し、E-boxエンハンサーを介してPer2遺伝子の転写を促進する新規の時計構成分子であると考えられた。また、ヒト・マウス・ラットからBmal2相同遺伝子をそれぞれ単離することができたことから、BMAL2は広く動物に存在する分子であることがわかった。【2】概日時計を構成する新規分子の単離に向けて、ディファレンシャルディスプレイ解析を行ったところ、概日時計の位相後退に伴って光誘導される遺伝子を単離することができた。RT-PCR解析の結果、この遺伝子は時刻特異的に光誘導されると共に概日時計によって発現が制御されていることがわかった。この遺伝子の全長cDNAを単離した結果、bZIPモチーフをもつ転写因子をコードしていることがわかった。【3】脳深部における光情報伝達機構を探るため、ハト脳深部に発現する光情報伝達分子の解析を進めた結果、光受容体ロドプシンとトランスデューシンが同一の細胞に共局在していることがわかった。さらに、先情報伝達経路の出力に位置する環状ヌクレオチド依存性カチオンチャンネルに特異的抗体を作成した

交付申請書に記載の研究実施計画に沿って研究を遂行し、下記の成果を得た。1.動物における光依存的転写調節に関わる遺伝子配列(光応答エレメント)は未だ同定されていない。そこで、初年度にひき続き、光に応答するピノプシン遺伝子のプロモータに着目し、ニワトリ松果体細胞を用いた転写アッセイによる解析を行った。その結果、18塩基からなる光応答エレメントを同定することに成功した。このエレメントには時刻情報の入力配列であるCACGTG型のE-box配列が含まれていたことから、松果体をはじめとする動物の光感受性時計細胞においては、光情報と時刻情報がE-box配列において統合されている可能性が強く示唆された。さらに、ゲルシフト解析の結果、この光応答エレメントに作用する因子は、肝臓等の種々の臓器に発現する転写抑制因子であることがわかった。光応答エレメントの解析と並行して、ニワトリ松果体において光応答するピノプシン以外の遺伝子を探索した結果、Hsp90遺伝子の転写が光刺激により顕著に増大することが判明した。2.新規時計関連分子の同定を目指して、時計発信系を内包するモデル培養細胞(rat-1細胞)を用い、時計遺伝子のリズム発現に関わる因子を探索した。その結果、rat-1細胞をグルコースにより刺激することによって、Per1をはじめとする時計遺伝子の発現量が一過的に減少し、それに続いて概日リズムを示すことを発見した。さらに、種々の薬剤を用いた解析の結果、グルコースによるリズム誘導減少には新たな遺伝子の転写が関与していることが示唆された。そこで次に、グルコースによるリズム誘導の分子メカニズムを知るための手がかりを得るために、マイクロアレイ解析によりグルコース応答遺伝子を検索した。その結果、時計遺伝子の発現を制御する分子の候補としてVdupおよびTieg1遺伝子を同定することができた

交付申請書に記載の研究目的に沿って概日リズムの発現と光位相シフトに関する研究を遂行し、以下の成果を得た。(1)ニワトリ松果体に発現する時計遺伝子Per2, Per3, Clock, Bmall及び新規のbHLH-PAS型転写因子Bmal2をクローニングした。機能解析の結果、BMAL2はCLOCKやBMAL1と結合し、転写の促進とその抑制という両方向性の転写調節機能を持つと推定された。また、ニワトリ松果体からCry1・Cry2遺伝子を単離し、培養松果体細胞でのmRNA量を明暗・恒暗条件下で調べた。その結果、両遺伝子の発現は概日時計のみならず光の制御を受けていることが判明し、Cry遺伝子が光位相シフトに関与する可能性が示唆された。(2)ニワトリ松果体において活性が日周変動を示すMAPキナーゼ(MAPK)は主観的暗期の光刺激により脱リン酸化(不活性化)されるが、この光応答現象がMAPKを脱リン酸化するフォスファターゼの光活性化に起因することを見出した。次に、時計細胞におけるMAPKの標的蛋白質を探索し、MAPKがBMAL1をリン酸化することを見出した。このリン酸化部位を決定し、そのうちThr534のリン酸化によりBMAL1の転写活性化能が抑制されることを明らかにした。これらの結果から、MAPKの活性リズムは概日時計の位相調節に重要な役割を果たしていると考えられた。(3)光位相シフトの分子機構を知るため、Differential Display法を用いてニワトリ松果体において時刻特異的に光誘導される遺伝子を網羅的に探索した。その結果、位相後退時に発現誘導されるbZIP型の転写因子cE4BP4を同定した。転写アッセイにおいてcE4BP4はcPer2の転写を抑制し、生体においてもcE4BP4はPer2の転写を光依存的に抑制して位相後退を誘導することから、光位相シフトの重要因子と考えられた

交付申請書に記載の研究実施計画に沿って研究を遂行し、下記の成果を得た。概日時計のリセットに関するタンパク質レベルからのアプローチとして、概日時計の前進時にニワトリの松果体において特異的に誘導される遺伝子を同定しA78と命名した。この遺伝子は、コイルドコイル構造を有しており、他のタンパク質と相互作用して機能を発揮する可能性が考えられた。そこでまず、このタンパク質の細胞内局在を調べた結果、中心体に局在していることがわかった。現在、中心体におけるA78の機能と中心体が概日時計発振(あるいは同調)に果たす役割を探るべく、中心体に局在するタンパク質とA78の相互作用を調べている。これと並行して、個体レベルからのリセットメカニズムの解明にむけて、これまでに作製したトランスジェニックゼブラフィッシュ(Tg)の遊泳行動解析を行った。その結果、このTgでは、個体内のメラトニンリズムが消失しているものの、かなりの割合の個体が遊泳行動のリズムを示すことが判明した。このことから、行動リズムを支配する中枢がメラトニンリズムの完全な支配下にあるわけではなく、体内のメラトニンリズムが消失しても、独自に発振できることが示唆された。一方、新たな入力分子の解析による時計同調の遺伝子プログラム解読を目指し、時計遺伝子Bmal1およびCry1のプロモータを単離した。現在、これらプロモータに光情報、グルコース刺激情報、あるいは時刻情報が入力している可能性を探っている

(1)Activation of Gil induced a phase-shift of the chick pineal circadian clock in a manner similar to that induced by light. In the promoter region of pinopsin gene, we identified a light-responsive element responsible for its light-dependent transcriptional activation.(2)Farnesylation of retinal rod G-protein transducin plays an important role in not only the cellular light-signaling but also the light-adaptation. We found that GRK7-1 expressed in the zebrafish retinal cones shows opsin-phosphorylating activity dozens-fold higher than that of rod-specific GRK1A.(3)Chick pineal and frog retinal MAPK was circadian-activated showing the peak activity at night. Within the mouse SCN, the profiles of the temporal and light activation of MAPK are both different between the dorsomedial and central regions of the SCN. We identified amino acid residues in BMAL1, CRY1 and CRY2 to be phosphorylated by MAPK and their phosphorylation-dependent functional changes. We also found that SCOP expressed abundantly in the SCN is a negative regulator of K-Ras and downstream MAPK. It was shown that p38 MAPK regulates the molecular clock in the daytime to have the phase-advancing effect.(4)When the chicks entrained to 12L : 12D conditions were exposed to prolonged light extending into the early night, pineal E4BP4 protein was kept at a high level, which delayed the morning induction of Per2 and delayed the phase of the pineal clock. E4BP4 was phosphorylated by CKIε, leading to its proteasomal degradation.(5)We found that glucose-stimulation of rat-1 cells resets the clock by a mechanism to which Tieg 1 and Vdup 1 contribute. TIEG 1 protein was in fact up-regulated upon glucose administration and it directly acts on the promoter of Bmall to inhibit the transcription.(6)Within the promoter region of pineal opsin exorhodopsin gene, we identified a cis-acting element, PIPE, that drives pineal-specific gene expression in the zebrafish

交付申請書に記載の研究実施計画に沿って研究を遂行し、下記の成果を得た。概日時計のリセットに関するタンパク質レベルからのアプローチとして、概日時計の前進時にニワトリの松果体において特異的に誘導される遺伝子を同定しA78と命名した。この遺伝子は、コイルドコイル構造を有しており、他のタンパク質と相互作用して機能を発揮する可能性が考えられた。そこでまず、このタンパク質の細胞内局在を調べた結果、中心体に局在していることがわかった。現在、中心体におけるA78の機能と中心体が概日時計発振(あるいは同調)に果たす役割を探るべく、中心体に局在するタンパク質とA78の相互作用を調べている。これと並行して、個体レベルからのリセットメカニズムの解明にむけて、これまでに作製したトランスジェニックゼブラフィッシュ(Tg)の遊泳行動解析を行った。その結果、このTgでは、個体内のメラトニンリズムが消失しているものの、かなりの割合の個体が遊泳行動のリズムを示すことが判明した。このことから、行動リズムを支配する中枢がメラトニンリズムの完全な支配下にあるわけではなく、体内のメラトニンリズムが消失しても、独自に発振できることが示唆された。一方、新たな入力分子の解析による時計同調の遺伝子プログラム解読を目指し、時計遺伝子Bmal1およびCry1のプロモータを単離した。現在、これらプロモータに光情報、グルコース刺激情報、あるいは時刻情報が入力している可能性を探っている

交付申請書に記載の研究実施計画に沿って研究を遂行し、下記の成果を得た。研究代表者らはこれまでに、概日時計の位相が前進する際にニワトリ松果体においてmRNAが誘導される新規分子を同定した。この分子をLCGと命名し、全長cDNAのクローニング・特異的抗体の作成ならびに強制発現系における細胞内局在の解析等を行ったところ、LCGは中心体のマーカーであるγチューブリンと共局在することが分かった。そこで次に、LCGが松果体においてγチューブリンと相互作用する可能性を免疫沈降法を用いて検討した。その結果、LCGとγチューブリンの結合が確認された。これらの結果から、LCGは時刻シグナルにしたがい中心体の活性を制御している分子である可能性が示唆された。LCGの解析と並行し、新しい光同調経路の発見を目指し、新規光受容分子を探索した。その結果、ニワトリゲノム中に、青色光受容タンパク質であるクリプトクロームの類似遺伝子が存在することを見いだした。これをcCRY4と命名し、cCRY4に関する解析を行った。その結果、cCRY4のmRNAは、網膜および松果体などの光感受性の概日時計組織に多く発現しており、培養した松果体細胞中においてmRNA量が光依存的に上昇することが判った。そこで、cCRY4を特異的に認識する抗体を作成し、タンパク質発現を調べたところ、松果体の多数の実質細胞および一部の間質細胞に存在することが判った。さらに、タンパク質発現もmRNAの発現量と同様の光依存的変動を示すことや、分画した細胞抽出液の水溶性画分中にcCRY4が検出されることがわかった。これらの結果から、cCRY4は光感受性の概日時計組織において青色光を受容する新規の光受容分子であり、ピノプシン等をはじめとする既知の光受容分子とは全く異なる光情報伝達経路に関与している可能性が強く示唆された

交付申請書に記載の研究計画に従い研究を遂行し、以下の知見を得た。【1】タンパク質レベルからの概日時計の発振プログラム解読これまでに同定した時計関連分子のうちA78およびE4BP4に重点をおき、蛋白質レベルでの解析を行った。その結果、A78は培養細胞中において中心体に局在することが判明した。概日時計の発振における中心体の役割は不明であるが、概日時計による細胞周期制御に関与する可能性が想定される。一方、E4BP4はニワトリ松果体において夕刻に限って転写が光誘導されるため、光位相後退に関わっている可能性を検討した。その結果、E4BP4蛋白質の発現に顕著なサーカディアンリズムが見られること、夕刻の光刺激によってmRNAレベルに加えて蛋白質量も増大することが判明した。さらに、時計関連キナーゼとして注目されているカゼインキナーゼによってE4BP4がリン酸化を受け、リン酸化によりプロテオソーム依存的に分解されることがわかった。これらの発見は、時計発振においてE4BP4が「いつ、どこで、何を」しているのかという時計発振プログラムの一端を明らかにしたものと言える。【2】個体レベルからの概日時計のリセットプログラム解読個体レベルにおける概日時計のリセットプロセスを解明するために、時計中枢の一つである松果体において時計機能を破壊したゼブラフィッシュの作製を試みた。まず、時計発振の中心的役割を担うBMAL分子のC末端領城を欠失した変異蛋白質を用い、これが時計発振に対してドミナントネガティブ効果を示すことを見いだした。次に、この変異BMAL蛋白質の遺伝子に松果体特異的プロモータを繋ぎ、松果体に変異BMALを発現するトランスジェニックゼブラフィッシュを作製した。その結果、変異遺伝子導入系統として複数のゼブラフィッシュ系統を樹立することに成功した

交付申請書に記載の研究実施計画に沿って研究を遂行し、下記の成果を得た。概日時計のリセットに関するタンパク質レベルからのアプローチとして、概日時計の前進時にニワトリの松果体において特異的に誘導される遺伝子を同定しA78と命名した。この遺伝子は、コイルドコイル構造を有しており、他のタンパク質と相互作用して機能を発揮する可能性が考えられた。そこでまず、このタンパク質の細胞内局在を調べた結果、中心体に局在していることがわかった。現在、中心体におけるA78の機能と中心体が概日時計発振(あるいは同調)に果たす役割を探るべく、中心体に局在するタンパク質とA78の相互作用を調べている。これと並行して、個体レベルからのリセットメカニズムの解明にむけて、これまでに作製したトランスジェニックゼブラフィッシュ(Tg)の遊泳行動解析を行った。その結果、このTgでは、個体内のメラトニンリズムが消失しているものの、かなりの割合の個体が遊泳行動のリズムを示すことが判明した。このことから、行動リズムを支配する中枢がメラトニンリズムの完全な支配下にあるわけではなく、体内のメラトニンリズムが消失しても、独自に発振できることが示唆された。一方、新たな入力分子の解析による時計同調の遺伝子プログラム解読を目指し、時計遺伝子Bmal1およびCry1のプロモータを単離した。現在、これらプロモータに光情報、グルコース刺激情報、あるいは時刻情報が入力している可能性を探っている

In order to utilize cryptochrome(CRY) as a molecular light switch for regulation of gene activity, we characterize CRYs in a variety of animals. Complementary DNA encoding cryptochromes were isolated from vertebrates such as Xenopus, quail, goldlined spinefoot. Because these CRYs are considered to interact with circadian clock components such as CLOCK and BMALs, we investigated whether the CRY proteins could interact with and inhibit the activities of those clock proteins by transcriptional analysis. Xenopus CRYs(XtCRY1 and XtCRY2) inhibited the transcriptional activities, suggesting that those CRYs work as circadian clock components in Xenopus. Next, we investigated mRNA expression of CRYs in the goldlined spinefoot, and observed that the CRY mRNA levels showed clear lunar rhythms with their peak at the first quarter moon. This strongly suggests that CRYs function for not only circadian clock but also lunar clock.Next, we searched putative CRY-interacting proteins by yeast two-hybrid screening. We identified several possible interactors, and interestingly, some of them showed light-dependent interaction, showing that they can be used as light-dependent switchable molecules. This system can be applied to regulation of gene activity by light at the transcription levels, and future experiments should be planned for light-dependent regulation of the protein localization, interaction, etc

クリプトクロム(CRY)は、広く生物界に存在する青色光受容分子であり、磁気受容能も含めて、組織特異的に多様な機能を担うと推定されている。CRYは、光センサーとして遺伝子発現調節や概日時計発振に関与し、また磁気受容分子としての機能も想定されるが、オプシン類に比べると機能解析が遅れている。これまでに、種々の動物においてCRY分子を多数同定し、特にニワトリCRY4に関しては最近、酵母を用いた発現・精製系を確立することに成功した。そこで本研究では、この系のさらなる改良を目指すと同時に、他のCRYタンパク質の精製法の確立、ならびに精製したCRY4を用いた分光学的解析ならびに生化学的解析により、光受容の分子機構を解析した。

本近年、動物のさまざまな網膜外組織に多様な光受容分子が存在することが明らかになってきた。このような光センサーのひとつとして特にクリプトクロムが注目を浴びつつある。クリプトクロムは、概日時計構成分子・青色光センサー・磁気センサーなどとして機能すると考えられているが、概日時計以外でのメカニズムはほとんど不明である。本研究では、研究代表者が最近得た知見に基づき、クリプトクロムが概月時計や磁気受容に関わる可能性を検証すると同時に、大量発現したクリプトクロムタンパク質の機能解析を通して、その光受容メカニズムを明らかにすることを目指した。具体的に本研究では、これまでの研究に基づき、【研究１】大量発現系を利用したタンパク質レベルでのCRY性状解析および【研究２】月齢時計の発振・同調におけるCRYの役割の解析を行った。それぞれの研究項目に関する成果は以下の通りである。【研究１】大量発現系を利用したタンパク質レベルでのCRY性状解析研究代表者はこれまでに、大腸菌をはじめ種々のタンパク質発現系を検討し、酵母を用いた系においてニワトリCRY4(cCRY4)を安定に発現させることに成功した。これまでに、温度感受性の抗cCRY4単クローン抗体を用いて温度変化のみでcCRY4を高純度精製し、スペクトル解析に供することに成功している（図１）。そこでこれをさらにスケールアップし、結晶構造解析に足る量のcCRY4を得る。【研究２】月齢時計の発振・同調におけるCRYの役割の解析概月時計の有無を知るために、自然条件下で採取していたゴマアイゴを稚魚段階で実験室に移動し長期間飼育した。月齢２周期以上にわたって間脳におけるSgCRY3のmRNA発現の解析によって、自然条件下で見られたSgCry3mRNAリズムが月光刺激の無い状況で持続するか等を調べた。その結果、SgCry3mRNAリズムは持続しなかったことから、実験室環境では月齢時計の発振は観察できなかった。一方、概月時計の局在を知るために、SgCRY3に対する抗体を作成し、飼育した魚の間脳におけるタンパク質の局在を調べたところ、興味深いことに、生殖応答に重要な間脳視床下部の乳頭部に存在する細胞群において顕著な発現が見られた。　以上の解析を通して、概月時計の発振を実験室で確認することはできなかったものの、月齢応答におけるCRY3の重要性を示すことができ、同時に、試験管内でのCRY解析にむけたタンパク質発現・精製系の確立に成功した。

本研究の申請段階において研究代表者らは、光受容分子CRYのC末端付近に対する１つのモノクローナル抗体(C13-mAb)が、抗原との結合において顕著な温度依存性を示すことを見いだしていた。この知見に基づき本研究では、温度依存的なタンパク質精製系を構築することを目指した基礎研究を行った。具体的には、申請書に記載した【研究１】および【研究２】を行った。なお、【研究３】モデルタンパク質へのタグ配列の付加と精製系としての実施例の提示は、進行中である。【研究１】C13-mAbが認識する抗原部位（エピトープ配列）を絞り込み　C13-mAbの作製に用いたcCRY4-CCT領域は60アミノ酸長である。モノクローナル抗体が特異的に認識するエピトープ配列は通常8-12アミノ酸長程度である。そこで、cCRY4-CCT領域を重複する約16アミノ酸長のペプチド10本に分割し、合成したペプチドをさまざまな濃度でC13-mAbと混合し、cCRY4-CCTを固定化した競合ELISAによりエピトープ部位を同定した。その結果、C13-mAbの抗原部位はCCT領域の最もC末端よりの13アミノ酸内に存在することが判明した。【研究２】エピトープ配列（単独および繰り返し配列）をN末あるいはC末に付加したモデルタンパク質の作製と免疫沈降・温度上昇による精製　上で決定した配列に基づき、エピトープ配列を付加したモデルタンパク質を大腸菌で発現させ、精製した。モデルタンパク質には、グルタチオンSトランスフェラーゼ(GST)を用いた。タグ配列は一般に、繰り返し配列とすることにより抗原と抗体の親和性が増大する可能性が考えられるので、抗原部位を２回および３回繰り返したGST融合タンパク質も同時に精製・解析した。その結果、GST融合タンパク質を含む大腸菌粗抽出物から、C13-mAb固定化レジンを用いて、低温でGST融合タンパク質を結合させ、温度上昇により溶出することに成功した。　以上の解析を通して、温度感受性の抗体が温和な条件におけるタンパク質の高純度精製に適したものとして利用可能であることを明確に示すことができたと確信している。

古くから、鳥類（ハト）をはじめ、魚類（サケ）・爬虫類（カメ）など幅広い動物が、地磁気情報を広範囲な移動に利用していることが示唆されてきた。近年、行動学的研究が数多く行われ、驚くべきことに、イモリやカエルといった広範囲の移動を伴わない生物にも地磁気を感じる能力があることが明らかになってきた。これはおそらく帰巣や定位に利用されていると考えられているが、そのメカニズムはまだほとんど不明である。光エネルギーを受けたクリプトクロム分子にラジカル対が生じ、ラジカル対が磁気効果を受けることによって、クリプトクロムが磁気センサーとして働くとするラジカルペア仮説が提唱されているが、そのメカニズムの詳細はほとんど不明である。脊椎動物の地磁気応答の分子機構の研究が遅れているのは、モデル脊椎生物において磁気的応答が見出されていないことが一因である。　このような背景の下、申請者らは、ゲノム配列が決定されておりモデル生物としての有用性が確立しているゼブラフィッシュに着目した。磁気応答性の有無を検討するため、種々の方法で予備的な解析を行った結果、ゼブラフィッシュの成魚が地磁気レベルの磁気に応答して定位行動を示すことを見いだした。そこで本研究ではさらに、この定位行動の詳細を解析した。地球上の緯度によって地磁気のベクトルは大きく異なっているため、実験室内で自在に地磁気の方向や強さを変化させられる地球磁場シミュレータを作成した。この装置を用いて、地磁気レベル磁気に対する応答性を調べたところ、(1)ゼブラフィッシュが一定の方位に向かうという選好方位を示すこと、(2)選好方位の方向は２方性であること、(3)選好方位の方向は動物集団によって異なること、(4)同一の親から生まれた集団は類似の方向を示す傾向があること、(5)方向性の有無は性や月齢あるいは飼育環境による影響を受けないこと、などを発見した。以上の結果は、ゼブラフィッシュが地磁気を感知して一定の方位を示すことを明確に示しており、モデル生物としての利点を生かすことによって、未知なる磁気受容の分子メカニズムに迫るための突破口が開かれたといえる。

　近年の研究から、多くの動物において網膜外のさまざまな組織に多様な光受容分子が存在し、外界の光を受容していることが明らかになってきた。このような光センサーのひとつとしてクリプトクロムが注目されている。昆虫では、クリプトクロムが青色光センサーとして機能していると同時に、光依存的な磁気センサーとしても機能すると考えられている。一方、脊椎動物では、概日時計に関わることは明らかになっているものの、光や磁気の受容に関与するかどうかは不明な点が多い。そこで本研究では、申請者がこれまで行ってきた光受容の研究に立脚しつつ、脊椎動物のクリプトクロムの光受容系と多様な機能の解明を目指した。　脊椎動物のクリプトクロムが概日時計以外の光応答現象に関わっている可能性を探るため、月齢に依存した産卵現象を示す熱帯性魚類であるゴマアイゴに着目した。まず、ゴマアイゴのクリプトクロム遺伝子を単離し、概日時計に関わる機能を調べたところ、概日時計の発振分子としての機能は見られなかった。そこで次に、月齢や性成熟との関連を調べたところ、産卵に伴う月齢応答に同期して脳の視床下部領域で発現変動していることを見いだした。このことからゴマアイゴのクリプトクロムは、概日時計よりむしろ月齢応答に関与していると考えられた。　これと並行して、青色光受容体候補分子としてのクリプトクロムの光受容メカニズムを探るために、培養細胞を用いたクリプトクロムの大量発現系の構築を試みた。大腸菌・バキュロウィルス・真核培養細胞等を検討したところ、真核細胞の一種である酵母において、ニワトリクリプトクロム４を高発現させることに成功した。この系を用いて発現したクリプトクロムは発色団であるFADと結合しており、光刺激に応じて吸収スペクトルの変化が観察された。このことから、光受容体としての活性を維持した状態で精製できたと考えられた。今後は、精製のためのタグの検討や、他のサブタイプのクリプトクロムの発現を進め、吸収スペクトル解析と同時に、レーザー分光やX線結晶構造解析、あるいは、相互作用タンパク質のプロテオーム解析を行う予定である。

　クリプトクロムは、動植物あるいはバクテリアにも存在する青色光受容体であり、光依存的な形態形成や概日リズムの調節などに働いている。ヒトをはじめとする哺乳類では、光受容体としては機能していない代わりに、概日リズムを制御する概日時計の構成分子として働いている。哺乳類以外の脊椎動物では、概日時計構成分子としての機能に加えて、光エネルギーを利用して地磁気を検知して方位や緯度を割り出すための磁気センサーとして機能する可能性が示唆されている。このようにクリプトクロムの多様な機能が明らかになりつつあるものの、概日時計の調節や磁気受容の分子機構はまだ謎につつまれている。そこで本研究では、さまざまな環境に棲息する脊椎動物を対象にクリプトクロム分子の発現や構造に関する研究を行った。具体的にはまず、両生類のモデル生物であるネッタイツメガエルにおいてクリプトクロム分子の解析を行った。その結果、ネッタイツメガエルにおいては３種類のクリプトクロム遺伝子が存在することがわかった。全身の主要な組織におけるmRNA発現を調べた結果、興味深いことに、いずれも卵巣において極めて高い発現が観察された。また、概日時計機能と深い関連がある月齢応答におけるクリプトクロムの機能を解析するために、月齢応答性の熱帯魚であるゴマアイゴにおけるクロプトクロムの解析も行った。その結果、ゴマアイゴより２種類のクリプトクロム遺伝子を同定することができ、いずれも、ゴマアイゴが産卵する上弦の月に脳において高い発現を示すことがわかった。ゴマアイゴクリプトクロムの発現パターンの解析より、月齢応答の分子機構モデルを提唱した。これらの研究と並行してさらに、光環境とクリプトクロム機能の関連を知るために、光がほとんど届かない深海に棲息するバラビクニンのクリプトクロムおよび時計遺伝子の解析を行った。その結果、バラビクニンのクリプトクロム遺伝子に加えて概日時計構成分子であるBMALおよびCLOCKの遺伝子も同定した。機能解析の結果、クリプトクロムは概日時計の発振を制御する転写調節機能を有することも明らかになった。これらの結果から、深海魚は特殊な環境に棲息するにも関わらず、概日時計を有することが強く示唆された。

近年の研究から、視細胞以外の動物組織に多様な光受容分子が存在し、外界の光を受容していることが明らかになってきた。このような光センサーのひとつとしてクリプトクロムが注目されている。昆虫では、クリプトクロムが青色光センサーとして機能していると同時に、光依存的な磁気センサーとしても機能する可能性が強く示唆されている。一方、脊椎動物にも複数のクリプトクロムが存在するものの、光や磁気の受容については全く不明である。我々はこれまで、鳥類をはじめ多数の生物においてクリプトクロムを同定し、概日時計機能を中心に解析を行ってきた。これまでの成果に基づきながら本研究では、脊椎動物クリプトクロムの新機能を探るためにアフリカツメガエルにおけるクリプトクロムの解析を行った。具体的にはまず、アフリカツメガエルに存在するクリプトクロム遺伝子の探索を行い、CRY1, CRY2, CRY4の３種類の遺伝子を同定した。次に、これらの遺伝子の発現部位を知るため、RT-PCR法を用いて組織ごとのCRYメッセンジャーRNAの定量を行った。その結果、興味深いことに、いずれのCRYも卵巣において、非常に高く発現していることが判った。これまでの研究では、CRYファミリー分子は、網膜や脳などの神経系、あるいは肝臓に高く発現していることが報告されていたが、こうした組織に比べても卵巣での発現は極めて高いことがわかった。次に、これら３種類のクリプトクロムの生理機能を探るため、転写アッセイを用いて、概日リズムの形成に関わる可能性を検討した。その結果、CRY1およびCRY2は、概日リズム形成の重要な鍵分子であるCLOCKおよびBMALに対して抑制的に働く転写抑制因子として機能することがわかった。一方、CRY4にはそのような機能は認められなかった。　以上の研究を通して、クリプトクロムが卵巣において何らかの機能を果たしていることが推定された。CRY1およびCRY2は特に、概日時計の発振か、あるいは何らかの転写調節に関与していると推定された。また、CRY4には転写制御機能は見られなかったことから、新奇の光受容体として機能している可能性が示唆された。

近年の研究から、脳や皮膚などの視細胞以外の部位にも光受容分子が存在し、外界の光を受容していることが明らかになってきた。これら網膜外光受容体には、レチナールを発色団とするオプシン型の光受容分子と、オプシンとは全く異なる光受容分子であるクリプトクロムがある。クリプトクロムは、青色感受性の光受容体として生物界に広く分布しており、動物においても光センサーとして機能していることが徐々に明らかになってきた。また、近年、伝書バトやイモリをはじめ多くの動物が光依存的に地磁場を感知して方位情報を探索や帰巣に役立てていることが証明され、その受容体としてもクリプトクロムが想定されている。このように、動物における新規の光応答経路と同時に磁場感知を担う分子として注目されているものの、クリプトクロムの研究は、オプシン類に比べるとかなり遅れている。このような背景の下に本研究では、クリプトクロムが光依存的な磁場センサーとして機能することを明らかにすることを目的として、広く脊椎動物のクリプトクロム遺伝子を単離・分析を行い、同時に皮膚細胞における光応答性を検討した。具体的にはまず、ニワトリCRY４と相互作用する分子の探索を行った。酵母ツーハイブリッド解析の結果、３種類の候補分子が単離され、それらをCRIP1-3と命名した。これらのうち、CRIP2分子については、抗体を作成して局在解析を行ったところ、網膜に特異的に発現していることがわかった。 [1]。また、NB1RGB細胞を用いたマイクロアレイ解析によりヒト皮膚細胞の光応答性を調べたところ、免疫応答性や食欲調節に関与するサイトカインであるMIC-1分子が青色光によって顕著に誘導されることを発見した[2]。

脊椎動物は、外界の光環境に応答するために多様な光センサーを持ち、脳や皮膚をはじめとして生体内の様々な部位において光に応答する。本研究では特に、動物の新規光センサー分子として着目されつつあるクリプトクロムおよびその光情報伝達経路の性状解析を行い、未知なる光情報制御機構を明らかにすることを目的とした。　具体的にはまず、網膜および松果体に発現する分子として、新規光受容体候補分子であるクリプトクロム４（CRY4）について、それらの遺伝子の単離・抗体作成・局在解析等を行った[1,2]。ニワトリCRY４に関しては、これまでに遺伝子の発現を調べていたため、モノクローナル抗体を作成し、まず生体における発現を調べた。その結果、網膜に特に高い発現が観察された。そこで次に、生体組織からのアフィニティー精製を行うために、エピトープ部位の決定と免疫沈降実験を行った。その結果、得られたモノクローナル抗体のうち少なくとも２種類が生体組織からの免疫沈降に使用可能であり、なかでも１種類では、エピトープペプチドによる蛋白質溶出が可能であった[1]。今後はこの抗体を用いて精製・単離したタンパク質複合体の解析によって、光情報伝達経路の全貌が明らかになると期待できる。これと並行して、個体レベルでのクリプトクロム解析の可能性を開拓するため、遺伝子操作が可能なゼブラフィッシュにおけるクリプトクロムの解析に着手した。まず、ゼブラフィッシュCRY４のカルボキシル末端付近を抗原としてモノクローナル抗体を作成したところ、抗原タンパク質に強く反応するモノクローナル抗体を複数単離することができた[2]。これらの鳥類や魚類における解析と並行して、ヒトの皮膚由来の初代培養繊維芽細胞を用いた光応答性の解析も行い、光に応答する遺伝子MIC-1を発見した[3]。　以上の解析を通して、クリプトクロムが介する新しい光情報伝達経路の解析に先鞭をつけることができた。今後は、今回作成した抗体を用いたアフィニティー精製産物の解析によって、CRY4が光情報を伝える因子の同定ができると期待される。

多くの脊椎動物は、外界の光環境に応答するために多様な光センサーを発達させている。特に、哺乳類以外の脊椎動物では、脳や皮膚をはじめとして生体内の様々な部位が光感受性を持つ。本研究では、動物の光センサー分子の探索と光情報伝達経路の性状解析を行い、未知なる光情報制御機構を明らかにするとともに、光センサーを用いた生体情報の制御に向けた研究を行った。　具体的にはまず、網膜および松果体に発現する分子として、新規光受容体候補分子であるクリプトクロム４（CRY4）および光情報伝達調節因子であるGRKについて、それらの遺伝子の単離・抗体作成・局在解析等を行った[1,2]。特にCRY4、これまでに知られている脊椎動物の光受容体（オプシン類）とは全く構造が異なり、植物や無脊椎動物にのみ知られている光受容分子ファミリーに属しており興味深い。また、鳥類の松果体には、ピノプシンやCRY4に加えて、メラノプシンが存在することも明らかにした[3]。鳥類の松果体は概日時計の中枢であることが知られているが、松果体や網膜以外にも卵巣にも独自の時計があり、組織特異的な機能に関与していることがわかった[4]。概日時計分子の機能解析としては、研究代表者らが以前発見した時計タンパク質であるBMAL2がPER2と結合して重要な働きをしていることをつきとめた[5]。これらの鳥類や魚類における解析と並行して、ヒトの皮膚由来の初代培養繊維芽細胞を用いた光応答性の解析も行い、光に応答する遺伝子MIC-1を発見した[6]。　以上の解析を通して、可視光に対して、脳・網膜・皮膚が応答する際に働く、新規の光受容タンパク質と光情報処理に関連する因子を明らかにすることができた。今後は、たとえばCRY4が光情報を伝える因子の同定やCRY４やメラノプシンの機能解析等を進める予定である。そうして得られる知見と今回得られた知見を総合することによって、光シグナルを利用して遺伝子発現等を制御することが可能になると期待できる。