2. Comparison of Wildtype vs. FASPS Mammalian Molecular Clocks By Erin Eppler May 2010 http://www.soundlighthealer.com/images/circadian_clock_1.jpg

3. A Typical Circadian Rhythm A circadian rhythm has a period of 24-25 hours, approximately one day’s length The master clock or suprachiasmatic nucleus (SCN) resides within the hypothalamus When light strikes specialized ganglion cells of the eye, an electrical impulse is sent to the SCN, accelerating the transcription of Per and Cry genes by CLOCK-Baml1 dimers, whose concentrations increase during the subjective night The cycle is a series of positive and negative feed back loops

4. Legend Baml1 gene Baml1 protein CLOCK gene CLOCK protein Per gene PER protein Cry gene CRY protein Ribosome Phosphorylating Unstable PER protein Cylin Kinase ε CKIε

5. During the subjective night concentrations of Baml1 and CLOCK proteins increase. Newly synthesized mRNA is transported from the nucleus to the cytoplasm where it is transcribed into Baml1 and CLOCK proteins. Cytoplasm Nucleus Baml1 Per 1 Ribosomes CLOCK Cry 1 Cry 2 Per 2 Per 3 CKIε

6. Baml1 and CLOCK protein dimerize and re-enter the nucleus where they bind to E-boxes on the promoter region of Per and Cry genes, accelerating transcription. Cytoplasm Nucleus Baml1 Per 1 Ribosomes CLOCK Cry 1 Cry 2 Per 2 Per 3 CKIε

7. Cytoplasm Nucleus Baml1 Per 1 Ribosomes CLOCK Cry 1 Cry 2 Per 2 Per 3 CKIε Movement of proteins out of the nucleus and into the cytoplasm

8. PER proteins that do not form a dimer are susceptible to phosphorylation by CKIε (Cyclin Kinase ε) making them less stable, leading to their degradation. Cytoplasm Nucleus Baml1 Per 1 Ribosomes CLOCK Cry 1 Cry 2 Per 2 Per 3 CKIε Phosphorylation

9. Cytoplasm Nucleus Baml1 Per 1 Ribosomes CLOCK Cry 1 Cry 2 Per 2 Per 3 CKIε Unstablization of PER proteins

10. CRY-PER or PER-PER dimers form in the cytoplasm and transport back into the nucleus. A CRY-PER dimer binds Baml1 and Cry genes, blocking transcription which leads to a negative feedback loop. Cytoplasm Nucleus Baml1 Per 1 Ribosomes CLOCK Cry 1 Cry 2 Per 2 Per 3 CKIε

11. Baml1 and CLOCK protein concentrations decrease over time. Cytoplasm Nucleus Baml1 Per 1 Ribosomes CLOCK Cry 1 Cry 2 Per 2 Per 3 CKIε

12. Degradation of BAM and CLOCK proteins will eventually prevent transcription of Per and Cry genes Cytoplasm Nucleus Baml1 Per 1 Ribosomes CLOCK Cry 1 Cry 2 Per 2 Per 3 CKIε

13. Cytoplasm Nucleus Baml1 Per 1 Ribosomes CLOCK Cry 1 Cry 2 Per 2 Per 3 CKIε

14. PER and CRY concentrations decrease and unblock Baml1 and CLOCK genes, thus restarting the cycle. Cytoplasm Nucleus Baml1 Per 1 Ribosomes CLOCK Cry 1 Cry 2 Per 2 Per 3 CKIε

15. Familial Sleep Phase Syndrome (FASPS) Molecular Clock Mutation occurs in the PER2 gene Circadian rhythm is advanced by 4-5 hours

16. The FASPS mutation causes accelerated nuclear clearance of PER2, but complex formation with CRY1 prevents nuclear export thereby causing nuclear accumulation and protein stabilization Cytoplasm Nucleus Baml1 Per 1 Ribosomes CLOCK Cry 1 Cry 2 Per 2 Per 3 CKIε