Reconstruction of circadian gene network by molecular LEGO

用“分子乐高”方法在果蝇S2细胞中重建了由人工嵌合基因组成的基因-蛋白质网络。模拟果蝇昼夜节律系统的双反馈环表现出一个相对稳定的周期为4-6小时的振荡。分析了对振荡的稳定性和周期有显著影响的参数，并对每个域中发生突变的成分进行了分析。负环成分信使核糖核酸半衰期的长短对振荡的稳定性有很大的影响。半衰期越短，节律性越稳定。然而，在负性因子中改变信使核糖核酸的半衰期并没有发现任何显著的周期变化。负因子在半衰期内2~16小时的蛋白质降解对振荡的稳定性和周期均无影响。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。负性因子的核局部化是产生周期性的一个重要参数，因为它的效率改变了重建节律的周期。此外，负性因子的核定位效率越低，就不会导致节律性。消极因素和积极因素之间的比例也很重要。当比例为1：5或5：1时，与它们为偶数时相比，一个给定的周期延长。令人惊讶的是，重建振荡在20到25摄氏度的范围内显示出温度补偿。

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.