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Analysis of the completed Drosophila genome has identified a gene of unknown function (encoded on bacterial artificial chromosomes AE003699 and AE003698) that is the structural homolog of mTim. A. L. Gotter, L. F. Kolakowski Jr., S. M. Reppert, unpublished data.
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Semiquantitative in situ hybridization with radioactive cRNA probes was performed as described (12).
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note
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The probe for Bmal1 was from nucleotides 864 to 1362 of mouse Bmal1b (GenBank accession number AF015203).
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note
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A breeding colony of mice carrying the Clk mutation on a BALB/c genetic background was established from mice provided by M. H. Vitaterna and J. S. Takahashi. Sex ratios of male and female mice were balanced across time points. Animal studies at Massachusetts General Hospital were approved by the Subcommittee on Research Animal Care.
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37
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0342631952
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note
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Brdm1 mutation was established on a C57BL/6 × 129SvEvBrd hybrid background (26). Balanced sex ratios of male and female mice were used.
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5) were seeded on glass cover slips in six-well dishes and transfected with Lipofectamine Plus (Gibco BRL) with 0.5 μg of total DNA per well. Forty-eight hours after transfection, cells were processed as described (13). A random population of 30 to 60 cells from each cover slip was examined by epifluorescence microscopy, and the subcellular distribution of expressed proteins was recorded without knowledge of treatment. At least three independently transfected cover slips were analyzed.
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note
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The mCry-deficient (double mutant) colony of mice had a C57BL/6 × 129 hybrid background, and wild-type controls were of the same genetic background (24). Sex ratios of male and female mice were balanced across time points.
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S2 cells were transfected with Cellfectin (Gibco BRL). Each transfection consisted of 10 to 100 ng of expression plasmid with indicated inserts in pAC5.1-V5, 10 ng of luciferase reporter, and 25 ng of β-galactosidase (β-Gal) internal control plasmid (driven by baculovirus immediate-early gene, ie-1 promoter). Total DNA for each transfection was normalized with pAC5.1-V5. Cells were harvested 48 hours after transfection. Luciferase activity was normalized by determining luciferase:β-Gal activity ratios and averaging the values from triplicate wells.
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MOP4 is a basic helix-loop-helix, PAS-containing transcription factor that is closely related to CLOCK. It can also heterodimerize with BMAL1 to activate transcription through E box enhancers (10, 13).
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50
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We thank S. Kay and C. Weitz for expression and reporter constructs and A. Chavda and C. Capodice for technical support. Supported by R37 HD14427 and RO1 NS39303, BBSRC 8/S09882, and a Spinoza Premium of the Netherlands Organization for Scientific Research. L.P.S. was supported in part by NIH Training Grant HL07901, K.K. by the University of Tokyo, and C.C.L. by the NIH.
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