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

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Presentation transcript:

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

Motivation

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

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

Proposed experiment Dissociation conditions Follow mRNA and protein levels Gonyaulax polyedra

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

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)

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

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