The Circadian Expression Of Gnb2l1

ObjectsOn the basis of our former experiments, the protein encoded by Gnb211 gene can interact with PERIOD1 protein, a crucial protein in circadian clock. So we investigate the circadian rhythm of Gnb211 gene and it's protein in vitro and in vivo in rat. There are two parts in the research: (1) investigate the expression level of circadian gene in C6 glioma cells by searching for a novel rhythm-inductor in vitro; then observe whether Gnb211 gene oscillates in a circadian manner and shows an immediately early expression under the stimulation in C6 glioma cells. (2) investigate the Circadian rhythm of Gnb211 gene and its protein in rat.MethodExperiment 1: To compare the induction efficiency of PMA with horse serum, the cultured NIH-3T3 cells were stimulated by PMA (phorbol 12-myristate 13-acetate, 100nM) and 50% horse serum respectively and performed RT-PCR to examine the expression of mPer1 mRNAs at the indicated times. The cultured C6 glioma cells were also stimulated by PMA and examined the expression of rPer1, rDbp mRNAs at the indicated times by RT-PCR. Then we examined expression level of rGnb211 mRNAs in C6 glioma cells at the different times.Experiment 2: Detecting Gnb211 mRNA level with the rat brain and liver samples by RT-PCR under condition of 12D/12L. Observing the expresssion level of RACK1 protein, which was encoded by Gnb211 gene, with the rat brain and liver samples by Western Blot under the same condition.ResultExperiment 1: Both PMA and horse serum could induce the circadian gene expression of mPer1 in NIH-3T3 cells and achieved similar induction efficiency, and it was confirmed that the circadian oscillation of rPer1, rDbp existed in C6 glioma cells. Based on former experiment, we performed the further research and found that Gnb211 mRNA displayed circadian oscillation in C6 glioma cells which was stimulated by PMA, and displayed an immediately early expression as Per1 did.Experiment 2: Gnb211 mRNA levels showed circadian oscillating in rat brain and liver under condition of 12D/12L. The protein, which was encoded by Gnb211 gene, also took on a circadian profile.ConclusionPMA is an effective rhythm inductor in vitro, and elicits the circadian oscillation of clock genes in C6 glioma cells. Gnb211 gene oscillates in a circadian manner in C6 cells, and undoubtedely is an important immediate early gene. Gnb211 gene and its protein also take on a circadian profile in rat. So, Gnb211 gene involves in the circadian molecular loop, and may play a crucial role in it.