Biyolojik saat genlerinde bulunan SNPlerin biyolojik saat üzerine etkilerinin moleküler düzeyde incelenmesi

The circadian clock is a biological rhythm most organisms exhibit oscillating with a period of around 24 hours, coordinated with the day and night cycle of the earth. This biological rhythm produces a daily oscillation of physiological, mental and behavioral states. The circadian clock is based on a complex molecular mechanism of transcriptional and translational feedback loops. The direct effect of this rhythm on the physiological and behavioral states is of great interest, whereas a disorder in molecular clock of mammalians has been shown to be associated with many diseases including but not limited to diabetes, cancer, schizophrenia, metabolic syndrome, autism and depression. Understanding the molecular mechanism of clock further may shed a light on the causes of these diseases and their treatments. Four core clock proteins generate rhythmicity in mammals. BMAL1 and CLOCK act as transcriptional activators, binding E-box elements on DNA and activating the expression of clock controlled genes, whereas CRYs and PERs repress BMAL1 and CLOCK proteins lead to a decrease in transactivation mediated by BMAL1:CLOCK heterodimer in clock controlled gene expression. SNPs in these critical clock genes may give rise to the molecular clock mechanism to dysfunction leading to the diseases associated with the circadian clock. In this thesis, SNPs of Bmal1, Clock and Cry1 genes were selected from the 1000 Genome Project to evaluate their effects on circadian rhythmicity using both molecular and biochemical approaches. The SNPs are located on the functional domains of the proteins and were selected based on their SIFT (Sorts Intolerant From Tolerant) and PolyPhen (Polymorphism Phenotyping) scores. The effects of mutations on BMAL1 and CLOCK proteins were tested by measuring the transactivation capacities on mPER1 promoter while the effect of mutations on CRY1 were measured by investigating any significant decrease on BMAL1:CLOCK transactivation capacity on mPER1 promoter. It is found that the Arg84Cys BMAL1 mutant located within the bHLH domain, result in decreased transactivation, whereas the Phe122Ser CLOCK, located within the bHLH domain result in increased transactivation of the E-box mediated gene transcription. 3 variants of CRY1, Gly144Val, Arg293His and Arg348Cys show reduced repression activity on BMAL1:CLOCK driven transcription. To see the effect of the mutant proteins on circadian rhythm lentivirus harboring appropriated wild type and mutant cDNA were infected into the U2OS cell line. Then circadian rhythms were measured by luminometric assay. Arg84Cys variant of Bmal1 overexpression showed a different oscillation than the wild type when overexpressed. Cry1 wild type, Gly144Val and Arg348Cys gave arrhythmic phenotypes when overexpressed, whereas Arg293His did not have any effect on the oscillation. Phe122Ser CLOCK mutant overexpression caused a slight increase in period length compared with wild type overexpression. In this thesis we are showing five core circadian clock gene alleles to have significant effects on biological rhythmicity. As many metabolic events and chronic disorders are related with the circadian mechanism, we propose that in the future these alleles may prove to be associated with disorders in humans, helping with their diagnosis and treatments.