THE USE OF A REVERSIBLE TRANSCRIPTION INHIBITOR, DRB, TO INVESTIGATE THE INVOLVEMENT OF SPECIFIC PROTEINS IN THE OCULAR CIRCADIAN SYSTEM OFAPLYSIA

Previously, the effects of 2-h treatments with the reversible transcri ption inhibitor 5,6-dichloro-1-beta-D-ribobenzimidazole (DRB) on the p hase of the circadian rhythm in the eye of Aplysia californica were st udied. Here we report a study of the effects of DRB on protein synthes is and a more detailed investigation of the effects of DRB on the phas e of the circadian rhythm. Treatments of DRB for 30 min reduced the ra te of transcription to about 30% of control values, and this inhibitio n reversed completely within 2 h after the end of the treatment. A pha se-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 obta ined 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 maxim um 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 o f the rhythm appeared more slowly (similar to 10 h) after the treatmen ts given during CT 6-12. Because the effects of DRB on the phase of th e overt rhythm appear to be rapid at a particular phase, it is very li kely that DRB affects the phase of the rhythm by altering the synthesi s of proteins during or shortly after the treatment. Thus we searched for proteins whose synthesis was altered by DRB. Incorporation of labe led 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 prot eins are worthy of further study as possible candidates for components of the circadian oscillator.