1. One oscillator or two? Understanding the oscillation that drives circadian rythmicity

2. Motivation

3. Rhythmic Dissociation in Gonyaulax Period of time in dim red conditions, with a 3 minute pulse of light every 30 minutes Two of the rhythms split: aggregation maintained a rhythm with τ = 21 hours and bioluminescence maintained a rhythm with τ = 24.6 hours (Roenneberg and Morse, 1993) Glow/flashing

4. Hypothesis There are (at least) two cellular oscillators driving circadian rhythmicity. Roenneberg: A, B Morse, Markovic, Roenneberg

5. Proposed experiment Dissociation conditions Follow mRNA and protein levels Gonyaulax polyedra

6. mRNA Luciferase and luciferase binding protein Okamoto, Hastings: 3% of unique genes exhibit circadian oscillations in transcript abundance (Procystis lunula Schütt) Rossini, Taylor, Fagan, Hastings: Lingulodinium polyedra –Thiolutin to block polymerase II –Half-life measured in hours In Gonyaulax: cDNA array to follow rythmicity

7. Protein Synthesis levels –35 S methionine incorporation in vivo (Markovic, Roenneberg, Morse) –Varied, without global change in protein levels Expression levels –2D gel analysis (Milos, et al)

8. Data analysis Do the mRNA levels, the protein expression, and cellular protein level rhythms follow aggregation, bioluminescence, or both? Both: proof of multiple oscillators One: proof of a single oscillator

9. Acknowledgments Dr. J. Woodland Hastings Dr. Charles Czeisler Dr. Joshua Gooley Members of MCB186