Reconstruction of circadian gene network by molecular LEGO

利用“分子乐高”方法在果蝇S2细胞中重构了人工嵌合基因组成的基因-蛋白网络。模拟果蝇昼夜节律系统的双反馈回路显示出相对稳定的振荡，周期为4 ~ 6小时。分析了对振荡稳定性和周期有显著影响的参数，各域的变化分量均有突变。负环组分mRNA的半衰期（以下称为负因子）对振荡的稳定性有显著影响。半衰期越短，节律性越稳定。然而，在负因子中改变mRNA的半衰期没有发现任何显著的周期变化。在半衰期2至16小时的负因子范围内的蛋白质降解对振荡的稳定性和周期都没有影响。当负因子的蛋白质半衰期与正因子的半衰期相似时，振荡变得稳定。发现负因子的核局部化是产生周期性的重要参数，因为它的效率改变了重构节奏的周期。此外，负因子的核定位效率较低，导致无节律性。正负因子的比值也很重要。当比例为1:5或5:1时延长的一段时间。令人惊讶的是，重建振荡在20 ~ 25℃范围内表现出温度补偿。

Gene-protein networks composed of artificial chimeric genes were reconstructed by "molecular LEGO" method in Drosophila S2 cell. A double feedback loop mimicking Drosophila circadian system showed a relatively stable oscillation with 4-6 hrs in period. Parameters which significantly influences to stability and period of the oscillation were analyzed with the altered components having mutation in each domain. The half life of mRNA of a negative loop component, hereafter called as a negative factor, drastically affects to a stability of oscillation. When the half-life becomes shorter, the rhythmicity becomes more stable. However, any significant change in period was not found by changing the half-life of mRNA in a negative factor. The protein degradation of a negative factor in the range from 2 to 16 hrs in its half-life had no effect on either stability or period of the oscillation. When the protein half-life of a negative factor was similar to that of a positive one, the oscillation becomes stable. The nuclear localization of negative factor was found as an important parameter to generate a periodicity because its efficiency changed the period of the reconstructing rhythm. Moreover, the less efficiency of nuclear localization of a negative factor induced no rhythmicity. The ratio between negative and positive factor is also important. A given period lengthened when the ratio is 1:5 or 5:1 as compared to when they were even. Surprisingly, the reconstructing oscillation showed a temperature-compensation in its period in the range between 20 and 25 C.