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S M Reppert

Publications and source records attributed to S M Reppert.

At least 19 recordsLinked to original sources
PubMed1999

mCRY1 and mCRY2 are essential components of the negative limb of the circadian clock feedback loop.

We determined that two mouse cryptochrome genes, mCry1 and mCry2, act in the negative limb of the clock feedback loop. In cell lines, mPER proteins (alone or in combination) have modest effects on their cellular location and ability to inhibit CLOCK:BMAL1 -mediated transcription. This suggested cryptochrome involvement in the negative limb of the feedback loop. Indeed, mCry1 and mCry2 RNA levels are reduced in the central and peripheral clocks of Clock/Clock mutant mice. mCRY1 and mCRY2 are nuclear proteins that interact with each of the mPER proteins, translocate each mPER protein from cytoplasm to nucleus, and are rhythmically expressed in the suprachiasmatic circadian clock. Luciferase reporter gene assays show that mCRY1 or mCRY2 alone abrogates CLOCK:BMAL1-E box-mediated transcription. The mPER and mCRY proteins appear to inhibit the transcriptional complex differentially.

3T3 Cells↗
PubMed1999

Differential regulation of mPER1 and mTIM proteins in the mouse suprachiasmatic nuclei: new insights into a core clock mechanism.

Recent discoveries have identified a framework for the core circadian clock mechanism in mammals. Development of this framework has been based entirely on the expression patterns of so-called "clock genes" in the suprachiasmatic nuclei (SCN), the principal clock of mammals. We now provide data concerning the protein expression patterns of two of these genes, mPer1 and mTim. Our studies show that mPER1 and mTIM are nuclear antigens expressed in the SCN and extensively throughout the forebrain. Expression of mPER1 in the SCN was rhythmic under entrained conditions and with clear circadian cycling under free-running conditions. Expression of mPER1 elsewhere in the mouse forebrain was not rhythmic. In contrast to mPER1, mTIM expression in the SCN did not vary with time in mice housed in either a light/dark cycle or in constant dim red light. The phase relationship between mPer1 RNA and mPER1 cycles in the SCN is consistent with a negative feedback model of the mammalian clock. The invariant nature of nuclear mTIM in the SCN suggests that its participation in negative feedback occurs only after mPER1 has entered the nucleus, and that the abundance of mTIM is not regulated by the circadian clock or the light/dark cycle.

Animals↗
PubMed1999

Assignment of the melatonin-related receptor to human chromosome X (GPR50) and mouse chromosome X (Gpr50).

Recent efforts to clone further members of the melatonin receptor family have led to the identification of a novel G-protein-coupled receptor in human pituitary. This receptor, referred to as H9, is clearly related to high-affinity melatonin receptors yet unable to bind this hormone. We now report the cloning and expression of the cDNA encoding the H9 receptor in mice. The mouse clone encodes a protein of 591 amino acids that shares 74% amino acid identity with the human receptor and is unable to bind 2-[125I]iodomelatonin when transiently expressed in COS-7 cells. We also determined the chromosome loci of the human and mouse H9 receptor genes. Both genes were found to be X-linked: radiation hybrid mapping revealed that the human H9 gene (GPR50) is localized to Xq28. The mouse gene (Gpr50) was determined to lie in the proximal portion of chromosome X by means of interspecific backcross analysis. These loci might be relevant to genetically based neuroendocrine disorders.

Amino Acid Sequence↗
PubMed1999

A molecular mechanism regulating rhythmic output from the suprachiasmatic circadian clock.

We examined the transcriptional regulation of the clock-controlled arginine vasopressin gene in the suprachiasmatic nuclei (SCN). A core clock mechanism in mouse SCN appears to involve a transcriptional feedback loop in which CLOCK and BMAL1 are positive regulators and three mPeriod (mPer) genes are involved in negative feedback. We show that the RNA rhythm of each mPer gene is severely blunted in Clock/Clock mice. The vasopressin RNA rhythm is abolished in the SCN of Clock/Clock animals, leading to markedly decreased peptide levels. Luciferase reporter gene assays show that CLOCK-BMAL1 heterodimers act through an E box enhancer in the vasopressin gene to activate transcription; this activation can be inhibited by the mPER and mTIM proteins. These data indicate that the transcriptional machinery of the core clockwork directly regulates a clock-controlled output rhythm.

3T3 Cells↗
PubMed1999

Expression of basic helix-loop-helix/PAS genes in the mouse suprachiasmatic nucleus.

The suprachiasmatic nuclei contain a circadian clock that drives rhythmicity in physiology and behavior. In mice, mutation of the Clock gene produces abnormal circadian behavior [Vitaterna M. H. et al. (1994) Science 264, 715-725]. The Clock gene encodes a protein containing basic helix-loop-helix and PAS (PER-ARNT-SIM) domains [King D. P. et al. (1997) Cell 89, 641-653]. The PAS domain may be an important structural feature of a subset of genes involved in photoreception and circadian rhythmicity. The expression and regulation of messenger RNAs encoding eight members of the basic helix-loop-helix/PAS protein superfamily were examined by in situ hybridization. Six of the genes studied (aryl hydrocarbon receptor nuclear transporter, aryl hydrocarbon receptor nuclear transporter-2, Clock, endothelial PAS-containing protein, hypoxia-inducible factor-1alpha and steroid receptor coactivator-1) were expressed in the suprachiasmatic nucleus of adult and neonatal mice. No evidence for rhythmicity of expression was observed when comparing brains collected early in the subjective day (circadian time 3) with those collected early in subjective night (circadian time 15). Neuronal PAS-containing protein-1 messenger RNA was expressed in the suprachiasmatic nucleus of adult (but not neonatal) mice, and a low-amplitude rhythm of neuronal PAS-containing protein-1 gene expression was detected in the suprachiasmatic nucleus. Neuronal PAS-containing protein-2 messenger RNA was not detected in adult or neonatal suprachiasmatic nucleus. Exposure to light at night (30 or 180 min of light, beginning at circadian time 15) did not alter the expression of any of the genes studied. The expression of multiple members of the basic helix-loop-helix/PAS family in the suprachiasmatic nucleus suggests a rich array of potential interactions relevant to the regulation of the suprachiasmatic circadian clock.

Aging↗
PubMed1998

Three period homologs in mammals: differential light responses in the suprachiasmatic circadian clock and oscillating transcripts outside of brain.

We have cloned and characterized the mouse cDNA of a third mammalian homolog of the Drosophila period gene and designated it mPer3. The mPER3 protein shows approximately 37% amino acid identity with mPER1 and mPER2 proteins. The three mammalian PER proteins share several regions of sequence homology, and each contains a protein dimerization PAS domain. mPer3 RNA levels oscillate in the suprachiasmatic nuclei (SCN) and eyes. In the SCN, mPer3 RNA levels are not acutely altered by light exposure at different times during subjective night. This contrasts with the acute induction by light of mPer1 and mPer2 RNA levels during early and late subjective night. mPer3 is widely expressed in tissues outside of brain. In liver, skeletal muscle, and testis, mPer RNAs exhibit prominent, synchronous circadian oscillations. The results highlight the differential light responses among the three mammalian Per genes in the SCN and raise the possibility of circadian oscillators in mammals outside of brain and retina.

Animals↗
PubMed1998

Molecular analysis of mammalian timeless.

We cloned the mouse cDNA of a mammalian homolog of the Drosophila timeless (tim) gene and designated it mTim. The mTim protein shows five homologous regions with Drosophila TIM. mTim is weakly expressed in the suprachiasmatic nuclei (SCN) but exhibits robust expression in the hypophyseal pars tuberalis (PT). mTim RNA levels do not oscillate in the SCN nor are they acutely altered by light exposure during subjective night. mTim RNA is expressed at low levels in several peripheral tissues, including eyes, and is heavily expressed in spleen and testis. Yeast two-hybrid assays revealed an array of interactions between the various mPER proteins but no mPER-mTIM interactions. The data suggest that PER-PER interactions have replaced the function of PER-TIM dimers in the molecular workings of the mammalian circadian clock.

Amino Acid Sequence↗
PubMed1998

Brain control of embryonic circadian rhythms in the silkmoth Antheraea pernyi.

The clock protein PER is necessary for circadian control of egg-hatching behavior in the silkmoth Antheraea pernyi. Since the brain and midgut of the silkmoth embryo contain PER-positive cells, we examined the circadian clock potential of these embryonic tissues. Transplantation experiments indicate that the circadian clock controlling egg-hatching behavior resides in brain, and that a humoral factor mediates this circadian regulation. We also used ligation experiments on first instar larvae to show that the circadian control of PER movement into the nuclei of midgut epithelial cells is dependent on an intact (connected) brain. These results implicate a novel brain factor in the circadian regulation of egg-hatching behavior and provide further evidence for differing mechanisms of PER control among species.

Animals↗
PubMed1997

Cellular construction of a circadian clock: period determination in the suprachiasmatic nuclei.

The circadian clock in the suprachiasmatic nuclei is composed of multiple, single-cell circadian oscillators (clock cells). We now test the hypothesis that the circadian period in behavior is determined by the mean period that arises from the coupling of clock cells with diverse circadian periods. For these studies, we monitored firing rate rhythms of individual suprachiasmatic nuclei neurons on fixed multielectrode plates and exploited the altered circadian periods expressed by heterozygous and homozygous tau mutant hamsters. The results show that circadian period in the whole animal is determined by averaging widely dispersed periods of individual clock cells. The data also demonstrate that the tau mutation affects circadian function in a cell-autonomous manner.

Animals↗
PubMed1997

Molecular dissection of two distinct actions of melatonin on the suprachiasmatic circadian clock.

The pineal hormone melatonin elicits two effects on the suprachiasmatic nuclei (SCN): acute neuronal inhibition and phase-shifting. Melatonin evokes its biological effects through G protein-coupled receptors. Since the Mel1a melatonin receptor may transduce the major neurobiological actions of melatonin in mammals, we examined whether it mediates both melatonin effects on SCN function by using mice with targeted disruption of the Mel1a receptor. The Mel1a receptor accounts for all detectable, high affinity melatonin binding in mouse brain. Functionally, this receptor is necessary for the acute inhibitory action of melatonin on the SCN. Melatonin-induced phase shifts, however, are only modestly altered in the receptor-deficient mice; pertussis toxin still blocks melatonin-induced phase shifts in Mel1a receptor-deficient mice. The other melatonin receptor subtype, the Mel1b receptor, is expressed in mouse SCN, implicating it in the phase-shifting response. The results provide a molecular basis for two distinct, mechanistically separable effects of melatonin on SCN physiology.

Animals↗
PubMed1997

Two period homologs: circadian expression and photic regulation in the suprachiasmatic nuclei.

We have characterized a mammalian homolog of the Drosophila period gene and designated it Per2. The PER2 protein shows >40% amino acid identity to the protein of another mammalian per homolog (designated Per1) that was recently cloned and characterized. Both PER1 and PER2 proteins share several regions of homology with the Drosophila PER protein, including the protein dimerization PAS domain. Phylogenetic analysis supports the existence of a family of mammalian per genes. In the mouse, Per1 and Per2 RNA levels exhibit circadian rhythms in the SCN and eyes, sites of circadian clocks. Both Per1 and Per2 RNAs in the SCN are increased by light exposure during subjective night but not during subjective day. The results advance our knowledge of candidate clock elements in mammals.

Amino Acid Sequence↗
PubMed1997

Melatonin receptors: molecular biology of a new family of G protein-coupled receptors.

A family of high-affinity, G protein-coupled receptors for the pineal hormone melatonin has been cloned from vertebrates. These recombinant receptors exhibits similar affinity and pharmacological characteristics to each other and to endogenous receptors, as defined with the melatonin agonist 2-[125I]iodomelatonin (125I-Mel). Two mammalian melatonin receptor subtypes have been identified by molecular cloning studies. The mammalian Mel1a melatonin receptor is expressed in most sites containing 125I-Mel binding. This includes the hypothalamic suprachiasmatic nucleus and hypophyseal pars tuberalis, presumed sites of the circadian and some of the reproductive actions of melatonin, respectively. The mammalian Mel1b melatonin receptor is expressed in retina and brain and may mediate the reported effects of melatonin on retinal physiology in some mammals. A third receptor subtype, the Mel1c melatonin receptor, has been cloned from zebra fish, Xenopus, and chickens but not from mammals. Molecular cloning of a melatonin receptor family now makes possible gene targeting to precisely determine the physiological role(s) of each receptor subtype.

Amino Acid Sequence↗
PubMed1997

The Mel1a melatonin receptor is coupled to parallel signal transduction pathways.

The recent cloning of a family of high affinity melatonin receptors has provided us with a unique opportunity to define the signal transduction pathways used by these receptors. We have studied signaling through the human Mel1a receptor subtype by stable expression of receptor complementary DNA in NIH 3T3 cells. Our data indicate that the human Mel1a receptor is coupled to inhibition of forskolin-stimulated cAMP accumulation by a pertussis toxin-sensitive G protein. Although melatonin alone is without effect on phosphoinositide hydrolysis, it potentiates the effects of PGF2 alpha stimulation on phospholipase C activation. Melatonin potentiates arachidonate release stimulated by PGF2 alpha and by ionomycin. The effects of melatonin on arachidonate release are sensitive to inhibition of protein kinase C. They are independent of the effects of melatonin on cAMP and do not appear to involve activation of mitogen-activated protein kinase. The effects of melatonin on both phosphoinositide hydrolysis and arachidonate release are sensitive to pertussis toxin treatment. Thus, we show that the melatonin signal is transduced by parallel pathways involving inhibition of adenylyl cyclase and potentiation of phospholipase activation.

3T3 Cells↗
PubMed1996

The Mel1a melatonin receptor gene is expressed in human suprachiasmatic nuclei.

The pineal hormone melatonin influences circadian rhythmicity in many vertebrate species. The circadian effects of melatonin in humans have led to its use to treat jet lag and circadian-based sleep disorders. Melatonin is thought to influence circadian rhythmicity by acting in the suprachiasmatic nuclei (SCN). The recent cloning of two melatonin receptor subtypes with high affinity for melatonin allows molecular analysis of melatonin receptors in human SCN. We report that Mel1a receptor mRNA is detectable in neonatal human SCN by in situ hybridization. Mel1b and melatonin-related receptor mRNAs were not detected. The presence of Mel1a receptor mRNA in human SCN supports the hypothesis that the Mel1a receptor is responsible for the circadian effects of melatonin in humans.

Adult↗
PubMed1996

Molecular characterization of prothoracicotropic hormone (PTTH) from the giant silkmoth Antheraea pernyi: developmental appearance of PTTH-expressing cells and relationship to circadian clock cells in central brain.

Using a PCR strategy, we have cloned the cDNA for prothoracicotropic hormone (PTTH) from the giant silkmoth, Antheraea pernyi. The A. pernyi PTTH cDNA encodes a preprohormone of 221 amino acids that is 51 and 71% identical at the amino acid level with Bombyx mori and Samia cynthia ricini PTTHs, respectively. Bacterially expressed, recombinant A. pernyi PTTH stimulates adult development when injected into debrained pupae. PTTH protein (ca. 30 kDa by Western blot) and mRNA (ca. 0.9 kb by Northern blot) are expressed in brain. Immunocytochemistry and in situ hybridization show that PTTH protein and mRNA are colocalized in L-NSC III from Day 4 of embryogenesis through adult life, with little variation in either protein or mRNA levels at the various ecdyses. A pair of cells expressing immunoreactivity for the circadian clock protein PER is located in the same region as PTTH-expressing L-NSC III in A. pernyi brain. However, double-label immunocytochemical studies show that PTTH and PER are located in different cells. The close anatomical location between PTTH- and PER-expressing cells suggests routes of communication between these two cell populations that may be important for the circadian control of PTTH release.

Amino Acid Sequence↗
PubMed1996

Cloning of a melatonin-related receptor from human pituitary.

We have cloned an orphan G protein-coupled receptor from a human pituitary cDNA library using a probe generated by PCR. The cDNA, designated H9, encodes a protein of 613 amino acids that is 45% identical at the amino acid level to the recently cloned human Mel(1a) and Mel(1b) melatonin receptors. Structural analyses of the encoded protein and its gene, along with phylogenetic analysis, further show that H9 is closely related to the G protein-coupled melatonin receptor family. Unusual features of the protein encoded by H9 include a lack of N-linked glycosylation sites and a carboxyl tail >300 amino acids long. H9 transiently expressed in COS-1 cells did not bind [125I]melatonin or [3H]melatonin. H9 mRNA is expressed in hypothalamus and pituitary, suggesting that the encoded receptor and its natural ligand are involved in neuroendocrine function.

Amino Acid Sequence↗