Reconstruction of circadian gene network by molecular LEGO

利用分子乐高方法在果蝇S2细胞中构建了人工嵌合基因的基因-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，并对各区域发生突变的变化成分进行了分析。负环成分的mRNA的半衰期（以下称为负因子）对振荡的稳定性有显著影响。当半衰期变短时，节律性变得更稳定。然而，通过改变mRNA的半衰期，在一个负的因素，没有发现任何显着的变化，在周期。负因子在其半衰期内2 - 16小时范围内的蛋白质降解对振荡的稳定性或周期均无影响。当负因子的蛋白质半衰期与正因子的蛋白质半衰期相近时，振荡变得稳定。负因子的核定位被认为是产生周期性的重要参数，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较低，不诱导节律性。消极因素和积极因素之间的比例也很重要。当比率为1：5或5：1时，与它们相等时相比，给定的周期延长。令人惊讶的是，重建振荡在其20和25 ℃之间的范围内的周期中显示出温度补偿。

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.