The Use of a Reversible Transcription Inhibitor, DRB, to Investigate the Involvement of Specific Proteins in the Ocular Circadian System of Aplysia

Abstract

Previously, the effects of 2-h treatments with the reversible transcription inhibitor 5,6-dichloro-1-β-D-ribobenzimidazole (DRB) on the phase of the circadian rhythm in the eye of Aplysia californica were studied. Here we report a study of the effects of DRB on protein synthesis and a more detailed investigation of the effects of DRB on the phase of the circadian rhythm. Treatments of DRB for 30 min reduced the rate of transcription to about 30% of control values, and this inhibition reversed completely within 2 h after the end of the treatment. A phase-response curve was obtained for 30-min treatments of DRB. Shorter (30 min) treatments with DRB produced phase shifts comparable to those produced by treatments with DRB for 2 h. The phase-response curve obtained using 30-min treatments of DRB was similar to one obtained using 2-h treatments with respect to the phase at which DRB exerts its maximum effect on the rhythm (around circadian time [CT] 6). However, some aspects of the two phase-response curves were different. The effect of DRB on the phase of the rhythm appeared rapidly after removal of DRB treatments given during CT 22-6, but the effects of DRB on the phase of the rhythm appeared more slowly (∼10 h) after the treatments given during CT 6-12. Because the effects of DRB on the phase of the overt rhythm appear to be rapid at a particular phase, it is very likely that DRB affects the phase of the rhythm by altering the synthesis of proteins during or shortly after the treatment. Thus we searched for proteins whose synthesis was altered by DRB. Incorporation of labeled amino acids into 2 proteins was found to be altered during the DRB treatment, whereas 15 proteins were affected after the DRB treatment. Among the proteins affected during or shortly after the DRB treatment were four previously identified proteins affected by other treatments that can shift the phase of the eye circadian rhythm. These four proteins are worthy of further study as possible candidates for components of the circadian oscillator.