Use of a high stringency differential display screen for identification of retinal mRNAs 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 differential display. Xenopus retina contains an endogenous circadian clock located within the photoreceptor layer. The retinal block 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 identification of messages that exhibit rhythmic expression. Out of approx. 2000 mRNAs that we have screened to date, we have identified four candidates for clock-controlled mRNAs. Initial characterization of one of these PCR products shows that it recognizes a pair of mRNA bands on Northern blots that exhibit high amplitude rhythms. This pair of messages 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 layer within the retina. Identification of new messages that are under the control of the circadian clock has broad relevance in retinal physiology and provides an opportunity to gain insight into the molecular mechanism of vertebrate circadian control.