Analysis of a mammalian clock gene, DIAL,a mammalian homologue of the Drosophila period gene

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Many biochemical, physiological and behavioral processes such as hormonal secretion and sleeping cycles exhibit circadian rhythms. Circadian rhythms are driven by species specific and endogenous clocks, and entrained mainly by daily light-dark cycles.I have developed a new method for the screening of homologous genes (IMS-PCR method) from different biological species. With this method, a human and a mouse homologue of the Drosophila period gene (hPER1 and mPer1, respectively) were identified. An autonomous circadian oscillation of mPer1 transcription was observed in the SCN under both the light-dark(LD) and the continuous dark conditions (DD). The expression of mPer1 was high during the daytime and low at night. In addition, transcription of mPer1 in the SCN was induced immediately after a short light pulse even at night. These results indicated that the circadian expression and the transient induction of mPer1 were involved in the central mechanisms of the mammalian circadian clock. I … More n this study, the molecular mechanisms of the circadian expression of mPer1 were analyzed. First, the genomic sequences and the transcriptional initiation sites of hPER1 and mPer1 were determined. Comparisons between two promoter regions of approximately 5 kb revealed that six regions containing five E boxes were well conserved. An expression reporter of mPer1 was constructed by the ligation of the promoter region of mPer1 with the luciferase ORF.In a transient expression assay using the mouse NIH3T3 cells, the mPer1 reporter was induced by its transcription factors, Clock and Bmal1. The enhancer sequences in the mPer1 promoter required for the transcriptional activation were determined. Moreover, the mammalian homologues of the Drosophila timeless gene were identified by the searches of EST databases in conjunction with the screenings of a human and a mouse brain cDNA library. The circadian expression of the mouse Timeless gene (mTim) was not detected as in the case of the tim gene in Drosophila. However, mTim and mPer1 synergistically inhibited the expression of mPer1. These results indicated that the circadian expression of mPer1 was maintained by the transcriptional switch involving the activation by Clock and Bmal1 and the autonomous repression by Pen and Tim. Less