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1.
Genome Res ; 10(12): 1928-40, 2000 Dec.
Article in English | MEDLINE | ID: mdl-11116088

ABSTRACT

The Clock gene encodes a basic helix-loop-helix (bHLH)-PAS transcription factor that regulates circadian rhythms in mice. We previously cloned Clock in mouse and human using a battery of behavioral and molecular techniques, including shotgun sequencing of two bacterial artificial chromosome (BAC) clones. Here we report the finished sequence of a 204-kb region from mouse chromosome 5. This region contains the complete loci for the Clock and Tpardl (pFT27) genes, as well as the 3' partial locus of the Neuromedin U gene; sequence analysis also suggests the presence of two previously unidentified genes. In addition, we provide a comparative genomic sequence analysis with the syntenic region from human chromosome 4. Finally, a new BAC transgenic line indicates that the genomic region that is sufficient for rescue of the Clock mutant phenotype is no greater than 120 kb and tightly flanks the 3' end of the Clock gene.


Subject(s)
Physical Chromosome Mapping , Sequence Analysis, DNA , Trans-Activators/genetics , 3-Oxo-5-alpha-Steroid 4-Dehydrogenase/genetics , Animals , CLOCK Proteins , Carrier Proteins/genetics , Chromosomes, Artificial, Bacterial/genetics , Circadian Rhythm/genetics , Cloning, Molecular , Computational Biology , Genetic Markers , Humans , Mice , Molecular Chaperones , Molecular Sequence Data , Multigene Family , Nerve Tissue Proteins/genetics , Neuropeptides/genetics , Phenotype , Physical Chromosome Mapping/methods , Promoter Regions, Genetic/genetics , Sequence Analysis, DNA/methods , Software
2.
Cell ; 103(7): 1009-17, 2000 Dec 22.
Article in English | MEDLINE | ID: mdl-11163178

ABSTRACT

Circadian oscillations in mammalian physiology and behavior are regulated by an endogenous biological clock. Here we show that loss of the PAS protein MOP3 (also known as BMAL1) in mice results in immediate and complete loss of circadian rhythmicity in constant darkness. Additionally, locomotor activity in light-dark (LD) cycles is impaired and activity levels are reduced in Mop3-/- mice. Analysis of Period gene expression in the suprachiasmatic nucleus (SCN) indicates that these behavioral phenotypes arise from loss of circadian function at the molecular level. These results provide genetic evidence that MOP3 is the bona fide heterodimeric partner of mCLOCK. Furthermore, these data demonstrate that MOP3 is a nonredundant and essential component of the circadian pacemaker in mammals.


Subject(s)
Circadian Rhythm/physiology , DNA-Binding Proteins , Transcription Factors/genetics , Transcription Factors/metabolism , ARNTL Transcription Factors , Animals , Basic Helix-Loop-Helix Transcription Factors , Behavior, Animal , Cell Cycle Proteins , DNA Probes , Gene Expression/physiology , Mammals , Mice , Mice, Knockout , Molecular Sequence Data , Motor Activity , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Period Circadian Proteins , Phenotype , Suprachiasmatic Nucleus/chemistry , Suprachiasmatic Nucleus/physiology
3.
Curr Opin Genet Dev ; 8(5): 595-602, 1998 Oct.
Article in English | MEDLINE | ID: mdl-9794822

ABSTRACT

Much progress has been made during the past year in the molecular dissection of the circadian clock. Recently identified circadian genes in mouse, Drosophila, and cyanobacteria demonstrate the universal nature of negative feedback regulation as a circadian mechanism; furthermore, the mouse and Drosophila genes are structurally and functionally conserved. In addition, the discovery of brain-independent clocks promises to revolutionize the study of circadian biology.


Subject(s)
Circadian Rhythm/genetics , Gene Expression Regulation , Animals , Cyanobacteria/genetics , Cyanobacteria/physiology , Drosophila/physiology , Mammals , Mice , Neurospora/genetics , Neurospora/physiology
4.
Science ; 280(5369): 1564-9, 1998 Jun 05.
Article in English | MEDLINE | ID: mdl-9616112

ABSTRACT

The mouse Clock gene encodes a bHLH-PAS protein that regulates circadian rhythms and is related to transcription factors that act as heterodimers. Potential partners of CLOCK were isolated in a two-hybrid screen, and one, BMAL1, was coexpressed with CLOCK and PER1 at known circadian clock sites in brain and retina. CLOCK-BMAL1 heterodimers activated transcription from E-box elements, a type of transcription factor-binding site, found adjacent to the mouse per1 gene and from an identical E-box known to be important for per gene expression in Drosophila. Mutant CLOCK from the dominant-negative Clock allele and BMAL1 formed heterodimers that bound DNA but failed to activate transcription. Thus, CLOCK-BMAL1 heterodimers appear to drive the positive component of per transcriptional oscillations, which are thought to underlie circadian rhythmicity.


Subject(s)
Circadian Rhythm/physiology , Nuclear Proteins/genetics , Trans-Activators/metabolism , Transcription Factors/metabolism , Transcriptional Activation , ARNTL Transcription Factors , Animals , Basic Helix-Loop-Helix Transcription Factors , Biological Clocks , CLOCK Proteins , Cell Cycle Proteins , Circadian Rhythm/genetics , Cloning, Molecular , Cricetinae , DNA/metabolism , Dimerization , Feedback , Gene Expression , Helix-Loop-Helix Motifs , Male , Mesocricetus , Mice , Mutation , Nuclear Proteins/metabolism , Period Circadian Proteins , Promoter Regions, Genetic , Retina/metabolism , Suprachiasmatic Nucleus/metabolism , Trans-Activators/genetics , Transcription Factors/genetics
5.
J Biol Chem ; 272(32): 20063-9, 1997 Aug 08.
Article in English | MEDLINE | ID: mdl-9242678

ABSTRACT

A broad array of stressors induce ACTH release from the anterior pituitary, with consequent stimulation of the adrenal cortex and release of glucocorticoids critical for survival of the animal. ACTH stimulates adrenocortical gene expression in vivo and inhibits adrenocortical cell proliferation. Binding of ACTH to its G-protein-coupled receptor stimulates the production of cAMP and activation of the protein kinase A pathway. The stress-activated protein kinases (SAPKs) (or c-Jun N-terminal kinases) and the extracellular signal-regulated kinases (ERKs) are members of the mitogen-activated protein kinase family of serine/threonine kinases, which have recently been implicated in G-protein-coupled receptor intracellular signaling. The SAPKs are preferentially induced by osmotic stress and UV light, whereas the ERKs are preferentially induced by growth factors and proliferative signals in cultured cells. In these studies, ACTH stimulated SAPK activity 3-4-fold both in the adrenal cortex in vivo and in the Y1 adrenocortical cell line. 12-O-Tetradecanoylphorbol-13-acetate but not cAMP induced SAPK activity in Y1 cells. The isoquinolinesulfonamide inhibitors H-8 and H-89 blocked ACTH induction of SAPK activity at protein kinase C inhibitory doses but not at protein kinase A inhibitory doses. The calcium chelating agent EGTA inhibited ACTH-induced SAPK activity and the calcium ionophore A23187 induced SAPK activity 3-fold. In contrast with the induction of SAPK by ACTH, ERK activity was inhibited in the adrenal cortex in vivo and in Y1 adrenal cells. Together these findings suggest that ACTH induces SAPK activity through a PKC and Ca+2-dependent pathway. The induction of SAPK and inhibition of ERK by ACTH in vivo may preferentially regulate target genes involved in the adrenocortical stress responses in the whole animal.


Subject(s)
Adrenocorticotropic Hormone/pharmacology , Calcium-Calmodulin-Dependent Protein Kinases/biosynthesis , Mitogen-Activated Protein Kinases , Adrenal Cortex/drug effects , Adrenal Cortex/enzymology , Animals , Calcium/metabolism , Calcium-Calmodulin-Dependent Protein Kinases/metabolism , Cells, Cultured , Cyclic AMP/pharmacology , Enzyme Induction , Gene Expression/drug effects , Genes, Immediate-Early , JNK Mitogen-Activated Protein Kinases , Male , Mitogen-Activated Protein Kinase 1 , Mitogen-Activated Protein Kinase 3 , Promoter Regions, Genetic , Protein Kinase C/metabolism , Protein-Tyrosine Kinases/metabolism , Rats , Tumor Necrosis Factor-alpha/pharmacology
6.
Cell ; 89(4): 655-67, 1997 May 16.
Article in English | MEDLINE | ID: mdl-9160756

ABSTRACT

As a complementary approach to positional cloning, we used in vivo complementation with bacterial artificial chromosome (BAC) clones expressed in transgenic mice to identify the circadian Clock gene. A 140 kb BAC transgene completely rescued both the long period and the loss-of-rhythm phenotypes in Clock mutant mice. Analysis with overlapping BAC transgenes demonstrates that a large transcription unit spanning approximately 100,000 base pairs is the Clock gene and encodes a novel basic-helix-loop-helix-PAS domain protein. Overexpression of the Clock transgene can shorten period length beyond the wild-type range, which provides additional evidence that Clock is an integral component of the circadian pacemaking system. Taken together, these results provide a proof of principle that "cloning by rescue" is an efficient and definitive method in mice.


Subject(s)
Circadian Rhythm/genetics , Trans-Activators/genetics , Animals , Base Sequence , CLOCK Proteins , Chromosome Mapping , Chromosomes, Bacterial , Circadian Rhythm/physiology , Cloning, Molecular , DNA Primers/genetics , Female , Genetic Complementation Test , In Situ Hybridization , Male , Mice , Mice, Transgenic , Mutation , Phenotype , RNA, Messenger/genetics , RNA, Messenger/metabolism , Trans-Activators/physiology
7.
Cell ; 89(4): 641-53, 1997 May 16.
Article in English | MEDLINE | ID: mdl-9160755

ABSTRACT

We used positional cloning to identify the circadian Clock gene in mice. Clock is a large transcription unit with 24 exons spanning approximately 100,000 bp of DNA from which transcript classes of 7.5 and approximately 10 kb arise. Clock encodes a novel member of the bHLH-PAS family of transcription factors. In the Clock mutant allele, an A-->T nucleotide transversion in a splice donor site causes exon skipping and deletion of 51 amino acids in the CLOCK protein. Clock is a unique gene with known circadian function and with features predicting DNA binding, protein dimerization, and activation domains. CLOCK represents the second example of a PAS domain-containing clock protein (besides Drosophila PERIOD), which suggests that this motif may define an evolutionarily conserved feature of the circadian clock mechanism.


Subject(s)
Circadian Rhythm/genetics , Cloning, Molecular , Trans-Activators/genetics , Amino Acid Sequence , Animals , Base Sequence , CLOCK Proteins , Chick Embryo , Chromosome Mapping , Conserved Sequence , DNA Primers/genetics , DNA, Complementary/genetics , Dogs , Drosophila/genetics , Evolution, Molecular , Humans , Mice , Molecular Sequence Data , Mutation , RNA, Messenger/genetics , Sequence Homology, Amino Acid
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