USE OF A HIGH STRINGENCY DIFFERENTIAL DISPLAY SCREEN FOR IDENTIFICATION OF RETINAL MESSENGER-RNAS THAT ARE REGULATED BY A CIRCADIAN CLOCK

We report here the initiation of a systematic screen to identify clock -controlled mRNAs from the retina of Xenopus laevis using mRNA differe ntial display. Xenopus retina contains an endogenous circadian clock l ocated within the photoreceptor layer. The retinal clock controls many aspects of physiology, including gene transcription. This screen uses differential display, a PCR based procedure, to compare retinal mRNA populations at different times of day in constant darkness, for identi fication of messages that exhibit rhythmic expression. Out of approx. 2000 mRNAs that we have screened to date, we have identified four cand idates for clock-controlled mRNAs. Initial characterization of one of these PCR products shows that it recognizes a pair of mRNA bands on No rthern blots that exhibit high amplitude rhythms. This pair of message s is called RM1 and shows peak levels of expression in the subjective night. In situ hybridization shows that this clock-controlled message is specifically localized to the clock containing photoreceptor cell l ayer within the retina. Identification of new messages that are under the control of the circadian clock has broad relevance in retinal phys iology and provides an opportunity to gain insight into the molecular mechanism of vertebrate circadian control.