Miyazaki Koyomi, Mezaki Miho, Ishida Norio
Clock Cell Biology, National Institute of Advanced Industrial Science and Technology (AIST), IMCB 6-5, 1-1-1, Higashi, Tsukuba, 305-8566 Japan.
Novartis Found Symp. 2003;253:238-48; discussion 249.
The circadian expression in Drosophila of clock gene products, such as PER and TIM, is thought to be important for driving overt rhythms. The constitutive expression of per by the heat-shock or rhodopsin promoters restores rhythmicity of the null allele of per, suggesting that per mRNA cycling may not be required for protein cycling or for locomotor rhythms. Furthermore, the constitutive expression of tim mRNA also supports protein cycling and behavioural rhythms in tim mutant flies. Other reports have also shown that eliminating the oscillations of PER and TIM proteins by their over-expression abrogated circadian rhythmicity. These data indicate that the circadian rhythmic expression of PER and TIM proteins is also important like their rhythmic mRNA expression in Drosophila. To compare the molecular mechanism of circadian clocks in divergent species, we report here cloning circadian mRNA and protein expression profiling of human clock genes in normal human fibroblasts. Circadian oscillations of hPer1, hPer2, hPer3, hBMAL1 and hCry2 mRNA expression were observed in serum-stimulated normal human fibroblasts. The serum shock of human fibroblasts also caused daily oscillations in the amount and size of human PER proteins as was shown using our novel antibodies. Inhibitor studies indicate that phosphorylation and degradation of PER proteins is an important process in the human molecular clock.
果蝇中生物钟基因产物（如PER和TIM）的昼夜节律表达被认为对驱动明显的节律很重要。通过热休克或视紫红质启动子组成型表达per可恢复per无效等位基因的节律性，这表明蛋白质循环或运动节律可能不需要per mRNA循环。此外，tim mRNA的组成型表达也支持tim突变果蝇中的蛋白质循环和行为节律。其他报告也表明，通过过表达消除PER和TIM蛋白的振荡会消除昼夜节律。这些数据表明，PER和TIM蛋白的昼夜节律表达在果蝇中与其节律性mRNA表达一样重要。为了比较不同物种中生物钟的分子机制，我们在此报告克隆正常人成纤维细胞中人类生物钟基因的昼夜节律mRNA和蛋白质表达谱。在血清刺激的正常人成纤维细胞中观察到hPer1、hPer2、hPer3、hBMAL1和hCry2 mRNA表达的昼夜节律振荡。使用我们的新型抗体显示，人成纤维细胞的血清休克也会导致人PER蛋白的数量和大小出现每日振荡。抑制剂研究表明，PER蛋白的磷酸化和降解是人类分子生物钟中的一个重要过程。
