Stopping the circadian pacemaker with inhibitors of protein synthesis.

Abstract

The requirement for protein synthesis in the mechanism of a circadian pacemaker was investigated by using inhibitors of protein synthesis. Continuous treatment of the ocular circadian pacemaker ofthe mollusc Budagouddiana with anisomycin or cycloheximide substantially lengthened (up to 39 and 52 hr, respectively) the free-running period of the rhythm.To determine whether high concentrations of inhibitor could stop the pacemaker, long pulse treatments of various durations (up to 44 hr) were applied and the subsequent phase of the rhythm was assayed. The observed phases of the rhythm after the treatments were a function of the time of the end of the treatment pulse, but only for treatments which spanned subjective dawn. The results provide evidence that protein synthesis is required in a phase-dependent manner for motion of the circadian pacemaker to continue.While there is considerable experimental interest in biological timing, little is known about the cellular and subcellular events which are responsible for generating circadian periodicities. Given the ubiquitous role of proteins and enzymes in intracellular processes and systems, it is reasonable to hypothesize that intracellular clocks are built by using proteins-that the biochemical processes responsible for generating circadian timing intimately involve transcription and/or translation and/or proteins themselves (1).This hypothesis has received support from numerous and diverse studies. For example, mutations have been examined which show deficits or alterations in the physiology of the circadian pacemaker (reviewed in refs.

Genetic and Molecular Analysis of Circadian Rhythms

“…Chronic treatment with transcription inhibitors might be expected to stop such a clock at or near to subjective dawn, about 180 • out of phase with the expectation for a Drosophila-like clock. In Gonyaulax, Aplysia, Bulla, and hamsters, in addition to Neurospora, the sensitive phase for transcription and/or translation inhibitors is in the late night to early morning, and where examined the clocks are seen to stop near to subjective dawn (21,23,52,92,102). Albeit with the caveats stated above, these data strongly suggest that a morning/day phased clock of the type seen in Neurospora may be widespread phylogenetically.…”

“…Although data speaking to this question are relatively few, the nearly universal observation from organisms ranging from Gonyaulax (23), Neurospora (21), Aplysia (92), Bulla (52), and hamsters (102) that circadian clocks show time-of-day-specific sensitivity to inhibitors of transcription and/or translation (reviewed in 6, 92) may make strong predictions if interpreted correctly. There are abundant caveats to these data, but (a) if the effects of the inhibitors on the clock are really due to the inhibition of the respective processes and (b) if the phases in the clock cycle at which inhibitors cause phase shifts can be interpreted as the times when essential clock proteins must be synthesized, then one could infer when in the clock cycle the critical protein(s) was being made.…”

Calcium plays a central role in phase shifting the ocular circadian pacemaker of Aplysia

“…Unfortunately, at other phases (e.g. CT 18-24), protein synthesis inhibitors cause phase shifts in the ocular rhythm and protein synthesis may even be part of the basic oscillator mechanism (Rothman and Strumwasser 1976;Jacklet 1977Jacklet , 1980Lotshaw and Jacklet 1986;Yeung and Eskin 1988;Khalsa et al 1992). Thus, it is not clear whether protein synthesis is generally required to phase shift the ocular rhythm.…”

Non-transcriptional oscillators in circadian timekeeping

“…These experiments showed that rhythmicity relies on transcription and translation only in a phase-dependent manner [99,100]. These experiments were only recently repeated in the unicellular green alga O. tauri [5], for which transcription and translation were found to be dispensable during significant parts of the cycle.…”